Benzonorbornadiene derivatives and reactions thereof

ABSTRACT

A bioorthogonal molecule can include a molecule having a structure according to:wherein R1—R8 are independently selected from H, a substituted or unsubstituted C1-C4 alkyl or alkylene group, COOH, COOR9, COR9, CONR9R10, CN, CF3, and SO2R9, and where R9 and R10 are independently selected from H and a substituted or unsubstituted C1-C4 alkyl or alkylene group, with the proviso that one of R3—R8 comprises a leaving group, and wherein X is O, S, N, SO, SO2, SR+, Se, PO2-, or NRR′+, and where Rand R′ are independently selected from H or a substituted or unsubstituted C1-C4 alkyl or alkylene group.

RELATED APPLICATION(S)

This application is a continuation of U.S. Pat. Application No.15/929,012, filed on May 4, 2018, now issued as U.S. Pat. No.11,560,384, which claims the benefit of U.S. Provisional Application No.62/501,656, filed on May 4, 2017, and U.S. Provisional Application No.62/502,427, filed on May 5, 2017, each of which is incorporated hereinby reference.

BACKGROUND

Bioorthogonal chemistry generally refers to chemical reactions that canoccur in biological systems without interfering with native biochemicalprocesses. Bioorthogonal chemistry provides reactions that arecompatible with biomolecules, which facilitates the performance ofchemistry in living organisms. Biocompatible reaction development hasfocused primarily on transformations that link two molecules, as suchbioorthogonal ligation reactions have broad applicability inbioconjugation chemistry, materials science, and chemical biology. Suchreactions have further been used to localize drugs and imaging agents atsites of disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates a modified target molecule, in accordance with anexample of the present disclosure.

FIG. 1B illustrates a method of making a modified target molecule, inaccordance with an example of the present disclosure.

FIG. 1C illustrates a method of reconstituting a modified targetmolecule, in accordance with an example of the present disclosure.

FIG. 2 illustrates a method of activating an inactive enzyme, inaccordance with an example of the present disclosure.

FIG. 3 illustrates a method of controlling interactions between a targetmolecule and another molecule, in accordance with an example of thepresent disclosure.

FIG. 4 illustrates another method of controlling interactions between atarget molecule and another molecule, in accordance with an example ofthe present disclosure.

FIG. 5A illustrates a modified nucleic acid, in accordance with anexample of the present disclosure.

FIG. 5B illustrates a method of reconstituting a modified nucleic acid,in accordance with an example of the present disclosure.

FIG. 5C illustrates a method of controlling hybridization betweenindividual nucleic acids, in accordance with an example of the presentdisclosure.

FIG. 5D illustrates a method of controlling nucleic acid folding, inaccordance with an example of the present disclosure.

FIG. 5E illustrates a method of modifying a nucleic acid, in accordancewith an example of the present disclosure.

FIG. 5F illustrates a method of reconstituting a modified nucleic acid,in accordance with an example of the present disclosure.

FIG. 5G illustrates a method of facilitating dissociation of nucleicacids, in accordance with an example of the present disclosure.

FIG. 6A illustrates a bioorthogonal molecule and a releasing moleculeproximately bound to a target molecule, in accordance with an example ofthe present disclosure.

FIG. 6B illustrates a bioorthogonal molecule and a releasing moleculeproximately bound to a target nucleic acid, in accordance with anexample of the present disclosure.

FIG. 6C illustrates a bioorthogonal molecule and a releasing moleculehaving hybridized targeting moieties, in accordance with an example ofthe present disclosure.

FIG. 6D illustrates another bioorthogonal molecule and a releasingmolecule having hybridized targeting moieties, in accordance with anexample of the present disclosure.

FIG. 6E illustrates a bioorthogonal molecule and a releasing moleculeproximately bound to a target molecule, in accordance with an example ofthe present disclosure.

FIG. 6F illustrates a method of releasing a leaving group from abioorthogonal molecule, in accordance with an example of the presentdisclosure.

FIG. 7A illustrates a bioorthogonal molecule and a releasing moleculeproximately bound to a target molecule, in accordance with an example ofthe present disclosure.

FIG. 7B illustrates a bioorthogonal molecule and a releasing moleculeproximately bound to a target nucleic acid, in accordance with anexample of the present disclosure.

FIG. 7C illustrates a bioorthogonal molecule and a releasing moleculehaving hybridized targeting moieties, in accordance with an example ofthe present disclosure.

FIG. 7D illustrates another bioorthogonal molecule and a releasingmolecule having hybridized targeting moieties, in accordance with anexample of the present disclosure.

FIG. 7E illustrates a bioorthogonal molecule and a releasing moleculeproximately bound to a target molecule, in accordance with an example ofthe present disclosure.

FIG. 8A illustrates a carrier molecule having a bioorthogonal moleculeattached thereto, in accordance with an example of the presentdisclosure.

FIG. 8B illustrates a method of releasing a leaving group Z from acarrier molecule, in accordance with an example of the presentdisclosure.

FIG. 8C illustrates another method of releasing a leaving group Z from acarrier molecule, in accordance with an example of the presentdisclosure.

FIG. 9A illustrates a carrier molecule having a bioorthogonal moleculeattached thereto, in accordance with an example of the presentdisclosure.

FIG. 9B illustrates a method of retaining a leaving group Z on a carriermolecule, in accordance with an example of the present disclosure.

FIG. 9C illustrates another method of retaining a leaving group Z on acarrier molecule, in accordance with an example of the presentdisclosure.

FIG. 10A illustrates a method of releasing a leaving group Z from abioorthogonal molecule, in accordance with an example of the presentdisclosure.

FIG. 10B illustrates another method of releasing a leaving group Z froma bioorthogonal molecule, in accordance with an example of the presentdisclosure.

FIG. 10C illustrates a method of transferring a leaving group Z to acarrier molecule, in accordance with an example of the presentdisclosure.

FIG. 11A illustrates a method of releasing a leaving group Z from abioorthogonal molecule, in accordance with an example of the presentdisclosure.

FIG. 11B illustrates a method of transferring a leaving group Z to acarrier molecule, in accordance with an example of the presentdisclosure.

FIG. 12A illustrates a method of releasing a leaving group Z from acarrier molecule, in accordance with an example of the presentdisclosure.

FIG. 12B illustrates a method of retaining a leaving group Z on acarrier molecule, in accordance with an example of the presentdisclosure.

FIG. 13A illustrates a method of linking separate molecules together, inaccordance with an example of the present disclosure.

FIG. 13B illustrates a method of linking separate nucleic acidstogether, in accordance with an example of the present disclosure.

FIG. 13C illustrates a method of linking separatematerials/macromolecules together, in accordance with an example of thepresent disclosure.

FIG. 13D illustrates a method of controlling the affinity of separatemolecules for one another, in accordance with an example of the presentdisclosure.

FIG. 13E illustrates a method of controlling the activity of an enzyme,in accordance with an example of the present disclosure.

FIG. 13F illustrates a method of controlling the affinity of separatemolecules for one another, in accordance with an example of the presentdisclosure.

FIG. 13G illustrates a method of controlling the affinity of separatemolecules for one another, in accordance with an example of the presentdisclosure.

FIG. 13H illustrates a method of controlling the half-life of a targetmolecule, in accordance with an example of the present disclosure.

FIG. 13I illustrates a method of releasing macromolecules from amaterial, in accordance with an example of the present disclosure.

FIGS. 14A-14S illustrate individual reaction schemes for compounds ofinterest.

FIG. 15 illustrates a reaction scheme for various bioorthogonalcompounds, in accordance with examples of the present disclosure.

FIG. 16A illustrates a color change resulting from the reaction of 1 andDPTz (c(1) = c(DPTz) = 6 mM, 24 h, RT).

FIG. 16B illustrates an HPLC analysis of a reaction of 2 with DPTz (c(2)= 6 mM; c(DPTz) = 18 mM; T = 37° C.).

FIG. 16C illustrates an analysis of reaction between 1 and DPTz atdifferent time points. From left to right: DPPz, DPTz, pNA and 1(Indicated with arrows). The mobile phase A was 0.1% TFA in water andmobile phase B was acetonitrile. A gradient of 0-100% B ranging from1-15 min and 100%B ranging from 15-18 min was run at a flow rate of 4.0mL/min. Retention time for DPPz: 9.87-9.96 min; DPTz: 9.98-10.05 min;pNA: 11.71-11.81 min; 1: 14.78-14.80 min.

FIG. 16D illustrates an analysis of reaction of 2 and DPTz at differenttime points. From left to right: DPPz, DPTz, pNA and 2 (Indicated witharrows). The mobile phase A was 0.1% TFA in water and mobile phase B wasacetonitrile. A gradient of 0-100% B ranging from 1-15 min and 100%Branging from 15-18 min was run at a flow rate of 4.0 mL/min. Retentiontime for DPPz: 9.85-10.08 min; DPTz: 10.06-10.14 min; pNA: 11.68-11.76min; 2: 14.32-14.37 min.

FIG. 16E illustrates a release analysis of 3 with DPTz at different timepoints. From left to right: DPPz, DPTz, pNA and 3 (Indicated witharrows). The mobile phase A was 0.1% TFA in water and mobile phase B wasacetonitrile. A gradient of 0-100% B ranging from 1-15 min and 100%Branging from 15-18 min was run at a flow rate of 4.0 mL/min. Retentiontime for DPPz: 9.83-10.18 min; DPTz: 10.00-10.23 min; pNA: 11.64-11.87min; 3: 16.64-16.89 min.

FIG. 17A illustrates an ¹H NMR analysis of the time-dependent pNArelease from 2.

FIG. 17B depicts a quantification of the release of p-nitroaniline (pNA)from 7-aza/oxa-BNBD 1-3.

FIG. 17C depicts concentrations of starting material (2) and reactionproducts (DPPz, pNA) as a function of time.

FIG. 17D illustrates full spectrum monitoring the bioorthogonal releasereaction of 1 with DPTz in DMSO-d₆. Legend: 1:Δ; DPTz:□; pNA:●; DPPz:■;I3:▲. I.S: Internal standard.

FIG. 17E illustrates full spectrum monitoring the bioorthogonal releasereaction of 2 with DPTz in DMSO-d₆. Legend: 2: Δ; DPTz:□; pNA:●; DPPz:■. I.S: Internal standard.

FIG. 17F illustrates full spectrum monitoring the bioorthogonal releasereaction of 3 with DPTz in DMSO-d₆. Legend: 3:Δ; DPTz: □; pNA:●; DPPz:■. I.S: Internal standard.

FIG. 17G illustrates full spectrum monitoring the bioorthogonal releasereaction of 2 with DPTz in DMSO-d₆/D₂O (9:1, v/v). Legend: 2:Δ; DPTz:□;pNA:●; DPPz: ■. I.S: Internal standard.

FIG. 18 illustrates an HPLC analysis of the reaction of 5 with PEG-Tz inDMSO/PBS solution (1:1, v.v) at different time points. From left toright: Released Dox, PEG-Tz and Prodrug 5 (Indicated with arrows). Themobile phase A was 0.1% TFA in water and mobile phase B wasacetonitrile. A gradient of 0-75% B ranging from 1-15 min and 75%-100% Bfrom 15-18 min and 100%B ranging from 15-18 min was run at a flow rateof 4.0 mL/min. Retention time for Free Dox: 11.80-11.87 min; PEG-Tz:12.50-12.60; 5: 15.68-15.74 min.

FIG. 19A illustrates a cytotoxicity assay against lung cancer A549cells. The results are expressed as the mean ± standard deviation (n =3).

FIG. 19B illustrates a cytotoxicity assay against lung cancer A549cells. The results are expressed as the mean ± standard deviation (n =3).

FIG. 20A illustrates a representative spectrum of an HPLC analysis ofthe stability of 5 in human serum at 5 minutes. Prodrug 5 is indicatedwith arrow. The mobile phase A was 0.1% TFA in water and mobile phase Bwas acetonitrile. A gradient of 0-100% B ranging from 1-15 min and 100%Branging from 15-18 min was run at a flow rate of 4.0 mL/min. Retentiontime for 5: 13.18-13.33 min.

FIG. 20B illustrates a representative spectrum of an HPLC analysis ofthe stability of 5 in human serum at 6 hours. The mobile phase A was0.1% TFA in water and mobile phase B was acetonitrile. A gradient of0-100% B ranging from 1-15 min and 100%B ranging from 15-18 min was runat a flow rate of 4.0 mL/min. Retention time for 5: 13.18-13.33 min.

FIG. 20C illustrates a representative spectrum of an HPLC analysis ofthe stability of 5 in human serum at 24 hours. The mobile phase A was0.1% TFA in water and mobile phase B was acetonitrile. A gradient of0-100% B ranging from 1-15 min and 100%B ranging from 15-18 min was runat a flow rate of 4.0 mL/min. Retention time for 5: 13.18-13.33 min.

FIG. 20D illustrates a representative spectrum of an HPLC analysis ofthe stability of 5 in human serum at 48 hours. The mobile phase A was0.1% TFA in water and mobile phase B was acetonitrile. A gradient of0-100% B ranging from 1-15 min and 100%B ranging from 15-18 min was runat a flow rate of 4.0 mL/min. Retention time for 5: 13.18-13.33 min.

FIG. 21 depicts the stability of 2 in human serum as measured by HPLC atλ_(Abs) = 317 nm.

FIG. 22A depicts the second-order rate constant k₂ of the reaction withthe benzonorbornadiene derivative 1 and the tetrazine DPTz in DMSOdetermined from a plot of pseudo-first order k_(obs) versusconcentration of 1. The results are expressed as the mean ± standarddeviation (n = 3).

FIG. 22B depicts the second-order rate constant k₂ of the reaction withthe benzonorbornadiene derivative 2 and the tetrazine DPTz in DMSOdetermined from a plot of pseudo-first order k_(obs) versusconcentration of 2. The results are expressed as the mean ± standarddeviation (n = 3).

FIG. 22C depicts the second-order rate constant k₂ of the reaction withthe benzonorbornadiene derivative 3 and the tetrazine DPTz in DMSOdetermined from a plot of pseudo-first order k_(obs) versusconcentration of 3. The results are expressed as the mean ± standarddeviation (n = 3).

FIG. 22D depicts the second-order rate constant k₂ of the reaction withthe benzonorbornadiene derivative 4 and the tetrazine DPTz in DMSOdetermined from a plot of pseudo-first order k_(obs) versusconcentration of 4. The results are expressed as the mean ± standarddeviation (n = 3).

FIG. 22E depicts the second-order rate constant k₂ of the reaction withthe benzonorbornadiene derivative 1 and the tetrazine DPTz in 90%DMSO/H₂O determined from a plot of pseudo-first order k_(obs) versusconcentration of 1. The results are expressed as the mean ± standarddeviation (n = 3).

FIG. 22F depicts the second-order rate constant k₂ of the reaction withthe benzonorbornadiene derivative 2 and the tetrazine DPTz in 90%DMSO/H₂O determined from a plot of pseudo-first order k_(obs) versusconcentration of 2. The results are expressed as the mean ± standarddeviation (n = 3).

FIG. 22G depicts the second-order rate constant k₂ of the reaction withthe benzonorbornadiene derivative 3 and the tetrazine DPTz in 90%DMSO/H₂O determined from a plot of pseudo-first order k_(obs) versusconcentration of 3. The results are expressed as the mean ± standarddeviation (n = 3).

FIG. 22H depicts the second-order rate constant k₂ of the reaction withthe benzonorbornadiene derivative 1 and the tetrazine PEG-Tz in 90%DMSO/H₂O determined from a plot of pseudo-first order k_(obs) versusconcentration of 1. The results are expressed as the mean ± standarddeviation (n = 3).

FIG. 22I depicts the second-order rate constant k₂ of the reaction withthe benzonorbornadiene derivative 2 and the tetrazine PEG-Tz in 90%DMSO/H₂O determined from a plot of pseudo-first order k_(obs) versusconcentration of 2. The results are expressed as the mean ± standarddeviation (n = 3).

FIG. 22J depicts the second-order rate constant k₂ of the reaction withthe benzonorbornadiene derivative 2 and the tetrazine PEG-Tz in 60%DMSO/PBS determined from a plot of pseudo-first order k_(obs) versusconcentration of 2. The results are expressed as the mean ± standarddeviation (n = 3).

FIG. 22K depicts the second-order rate constant k₂ of the reaction withthe benzonorbornadiene derivative 1 and the tetrazine PEG-Tz in 60%DMSO/PBS determined from a plot of pseudo-first order k_(obs) versusconcentration of 1. The results are expressed as the mean ± standarddeviation (n = 3).

FIG. 23A depicts representative spectra of HPLC analysis of thestability of 5 in DMSO/PBS solution at 5 min. Prodrug 5 is indicatedwith arrow. The mobile phase A was 0.1% TFA in water and mobile phase Bwas acetonitrile. A gradient of 0-100% B ranging from 1-15 min and 100%Branging from 15-18 min was run at a flow rate of 4.0 mL/min. Retentiontime for 5: 13.16-13.20 min.

FIG. 23B depicts representative spectra of HPLC analysis of thestability of 5 in DMSO/PBS solution at 6 hrs. Prodrug 5 is indicatedwith arrow. The mobile phase A was 0.1% TFA in water and mobile phase Bwas acetonitrile. A gradient of 0-100% B ranging from 1-15 min and 100%Branging from 15-18 min was run at a flow rate of 4.0 mL/min. Retentiontime for 5: 13.16-13.20 min.

FIG. 23C depicts representative spectra of HPLC analysis of thestability of 5 in DMSO/PBS solution at 24 hrs. Prodrug 5 is indicatedwith arrow. The mobile phase A was 0.1% TFA in water and mobile phase Bwas acetonitrile. A gradient of 0-100% B ranging from 1-15 min and 100%Branging from 15-18 min was run at a flow rate of 4.0 mL/min. Retentiontime for 5: 13.16-13.20 min.

FIG. 24 illustrates product distributions as a function of time for thereaction of 1 and DPTz in DMSO-d₆/D₂O (9:1, v/v) at 37° C. Conditionswere the same as for ¹H NMR mechanism studies. The results are expressedas the mean ± standard deviation (n = 3).

These drawings are provided to illustrate various aspects of theinvention and are not intended to be limiting of the scope in terms ofdimensions, materials, configurations, arrangements or proportionsunless otherwise limited by the claims.

DETAILED DESCRIPTION

While these exemplary embodiments are described in sufficient detail toenable those skilled in the art to practice the invention, it should beunderstood that other embodiments may be realized and that variouschanges to the invention may be made without departing from the spiritand scope of the present invention. Thus, the following more detaileddescription of the embodiments of the present invention is not intendedto limit the scope of the invention, as claimed, but is presented forpurposes of illustration only and not limitation to describe thefeatures and characteristics of the present invention, to set forth thebest mode of operation of the invention, and to sufficiently enable oneskilled in the art to practice the invention. Accordingly, the scope ofthe present invention is to be defined solely by the appended claims.

Definitions

In describing and claiming the present invention, the followingterminology will be used.

As used herein, the term “about” is used to provide flexibility andimprecision associated with a given term, metric or value. The degree offlexibility for a particular variable can be readily determined by oneskilled in the art. However, unless otherwise enunciated, the term“about” generally connotes flexibility of less than 5% in some examples,less than 1% in other examples, and less than 0.01% in yet otherexamples.

As used herein with respect to an identified property or circumstance,“substantially” refers to a degree of deviation that is sufficientlysmall so as to not measurably detract from the identified property orcircumstance. The exact degree of deviation allowable may in some casesdepend on the specific context.

As used herein, “adjacent” refers to the proximity of two structures orelements. Particularly, elements that are identified as being “adjacent”may be either abutting or connected. Such elements may also be near orclose to each other without necessarily contacting each other. The exactdegree of proximity may in some cases depend on the specific context.

The term “dosage unit” or “dose” are understood to mean an amount of anactive agent that is suitable for administration to a subject in orderachieve or otherwise contribute to a therapeutic effect. In someexamples, a dosage unit can refer to a single dose that is capable ofbeing administered to a subject or patient, and that may be readilyhandled and packed, remaining as a physically and chemically stable unitdose.

As used herein, a “dosing regimen” or “regimen” such as “treatmentdosing regimen,” or a “prophylactic dosing regimen” refers to how, when,how much, and for how long a dose of an active agent or composition canor should be administered to a subject in order to achieve an intendedtreatment or effect.

As used herein, the terms “treat,” “treatment,” or “treating” refers toadministration of a therapeutic agent to subjects who are eitherasymptomatic or symptomatic. In other words, “treat,” “treatment,” or“treating” can be to reduce, ameliorate or eliminate symptoms associatedwith a condition present in a subject, or can be prophylactic, (i.e. toprevent or reduce the occurrence of the symptoms in a subject). Suchprophylactic treatment can also be referred to as prevention of thecondition.

As used herein, the terms “therapeutic agent,” “active agent,” and thelike can be used interchangeably and refer to an agent that can have abeneficial or positive effect on a subject when administered to thesubject in an appropriate or effective amount.

The phrase “effective amount,” “therapeutically effective amount,” or“therapeutically effective rate(s)” of an active ingredient refers to asubstantially non-toxic, but sufficient amount or delivery rates of theactive ingredient, to achieve therapeutic results in treating a diseaseor condition for which the drug is being delivered. It is understoodthat various biological factors may affect the ability of a substance toperform its intended task. Therefore, an “effective amount,”“therapeutically effective amount,” or “therapeutically effectiverate(s)” may be dependent in some instances on such biological factors.Further, while the achievement of therapeutic effects may be measured bya physician or other qualified medical person using evaluations known inthe art, it is recognized that individual variation and response totreatments may make the achievement of therapeutic effects a subjectivedecision. The determination of a therapeutically effective amount ordelivery rate is well within the ordinary skill in the art ofpharmaceutical sciences and medicine.

As used herein, “formulation” and “composition” can be usedinterchangeably and refer to a combination of at least two ingredients.In some embodiments, at least one ingredient may be an active agent orotherwise have properties that exert physiologic activity whenadministered to a subject. For example, amniotic fluid includes at leasttwo ingredients (e.g. water and electrolytes) and is itself acomposition or formulation.

As used herein, a “subject” refers to an animal. In one aspect theanimal may be a mammal. In another aspect, the mammal may be a human.

As used herein, a plurality of items, structural elements, compositionalelements, and/or materials may be presented in a common list forconvenience. However, these lists should be construed as though eachmember of the list is individually identified as a separate and uniquemember. Thus, no individual member of such list should be construed as ade facto equivalent of any other member of the same list solely based ontheir presentation in a common group without indications to thecontrary.

Concentrations, amounts, and other numerical data may be presentedherein in a range format. It is to be understood that such range formatis used merely for convenience and brevity, and should be interpretedflexibly to include not only the numerical values explicitly recited asthe limits of the range, but also to include all the individualnumerical values or sub-ranges encompassed within that range as if eachnumerical value and sub-range is explicitly recited. For example, anumerical range of about 1 to about 4.5 should be interpreted to includenot only the explicitly recited limits of 1 to about 4.5, but also toinclude individual numerals such as 2, 3, 4, and sub-ranges such as 1 to3, 2 to 4, etc. The same principle applies to ranges reciting only onenumerical value, such as “less than about 4.5,” which should beinterpreted to include all of the above-recited values and ranges.Further, such an interpretation should apply regardless of the breadthof the range or the characteristic being described.

Any steps recited in any method or process claims may be executed in anyorder and are not limited to the order presented in the claims.Means-plus-function or step-plus-function limitations will only beemployed where for a specific claim limitation all of the followingconditions are present in that limitation: a) “means for” or “step for”is expressly recited; and b) a corresponding function is expresslyrecited. The structure, material or acts that support the means-plusfunction are expressly recited in the description herein. Accordingly,the scope of the invention should be determined solely by the appendedclaims and their legal equivalents, rather than by the descriptions andexamples given herein.

Bioorthogonal Molecules, Compositions, and Reactions

Bioorthogonal chemistry provides biomolecule-compatible reactionscapable of being performed in living organisms. Biocompatible reactiondevelopment has focused primarily on transformations that link twomolecules, as such bioorthogonal ligation reactions have broadapplicability in bioconjugation chemistry, materials science, andchemical biology. Such reactions can be used to localize drugs andimaging agents at specific locations, such as sites of disease. Incontrast, the discovery of bioorthogonal cleavage reactions that allowfor the controlled release of payloads has only recently attractedsubstantial research interest, even though such reactions are valuablein a wide range of applications. For example, dissociative bioorthogonalreactions can be used in proximity-reporting analytical probes forsensing genetic markers and proteins in cells and in animals. Furtherapplications can include DNA sequencing, enzyme uncaging, cell imaging,biomacromolecule purification, multiplexed in situ protein detection,and as ultra-mild protecting groups, to name a few. Applications ofdissociative in vivo chemistry leading to spatiotemporally controlledrelease of drugs are particularly appealing because of the potential forclinical translation in cancer chemotherapy. For example, implantationof tetrazine (Tz)-modified biomaterials can facilitate the localizedactivation of prodrugs (e.g. doxorubicin), potentially providingsignificantly more potent anti-tumor effects relative to systemicdoxorubicin administration, which can also reduce side-effects. Inanother example approach, bioorthogonal reactions can be designed toactivate antibody-drug conjugates in vivo. In this pre-targetingstrategy, an antibody conjugated with a drug via a chemically-cleavablelinker can accumulate at a desired site, and the subsequentadministration of a trigger molecule can liberate the cytotoxic agentspecifically in the target tissue.

The scarcity of bioorthogonal bond-cleavage reactions, however, remainsa key bottleneck for the advancement of reaction-based applications inchemical biology and smart therapeutics. Until recently, modifiedStaudinger reactions were the only bioorthogonal release reactionsavailable. The development of the inverse-electron demand Diels-Alder(IEDDA) pyridazine elimination reaction between carbamate-modifiedtrans-cyclooctenes (TCO) and Tz was a breakthrough in this regard. Therate of this reaction was significantly faster than the Staudingerreaction, and it obviated the use of metabolically unstable phosphines,allowing for widespread use in chemical biology. Further examples ofbioorthogonal cleavage reactions included the strain-promoted1,3-dipolar cycloaddition of TCO and p-azidobenzylcarbamates, andTz-mediated removal of vinyl ethers. However, reactions need to meetseveral strict requirements for in vivo use, including rapid reactionrate, near-quantitative payload release, non-toxic reagents, andextended serum stability. Currently available reactions meet theseconditions only partially and further development will be necessary toachieve the full potential of in vivo drug activation.

Accordingly, the present disclosure describes bioorthogonal molecules(occasionally referred to herein as BNBD) that address many of theseconcerns. Generally, bioorthogonal molecules can have a structureaccording to Formula I:

where X is a heteroatom, such as oxygen, sulfur, (modified) nitrogen, orthe like, for example. R¹—R⁸ are individually selected from hydrogen ora substituted or unsubstituted C₁-C₄ alkyl or alkylene group, COOH,COOR⁹, COR⁹, CONR⁹R¹⁰, CN, CF₃, SO₂R⁹, or the like, with the provisothat one of R³—R⁸ comprises a leaving group. In some examples, R⁹ andR¹⁰ are independently selected from H and a substituted or unsubstitutedC₁-C₄ alkyl or alkylene group.

In further detail, X can include a variety of heteroatoms. Somenon-limiting examples of X can include O, S, N, N—Ac, N—Boc, SO, SO₂,SR⁺, Se, PO₂ ⁻, NRR′⁺, or the like. In some examples, R and R′ areindependently selected from H or a substituted or unsubstituted C₁-C₄alkyl or alkylene group. In some specific examples, X can include O orN, including N modified with acyl, alkyl, aryl, heteroaryl, andalkyoxycaronyl groups.

In some additional examples, R¹, R², or both can include anelectron-withdrawing group E. A variety of electron withdrawing groupscan be used in the bioorthogonal molecules, such as halides, amides,esters, carboxylic acids, acyl chlorides, ketones, aldehydes, amines,nitro groups, sulfonates, cyano groups, trihalides, the like, or acombination thereof. Non-limiting examples can include —COOH, —COOR⁹,—COR⁹, —CONR⁹R¹⁰, —CN, —CF₃, —SO₂R⁹, —NO₂, or the like. In someexamples, R⁹ and R¹⁰ are independently selected from H and a substitutedor unsubstituted C₁-C₄ alkyl or alkylene group. In some examples, R¹ caninclude an electron withdrawing group. In some additional examples, R²can include an electron withdrawing group. In yet other examples, bothR¹ and R² can include an electron withdrawing group. Thus, in someexamples, the bioorthogonal molecule can have a structure according toFormula II:

such as

for example, where Z is a leaving group.

A variety of leaving groups can be used in the present bioorthogonalmolecules. In some examples, the leaving group can be an A-Z group,where A is a linker group and Z is a leaving group. Where this is thecase, the linker group can generally include a C₁-C₃ alkyl or alkylenegroup, although a variety of other linker groups may also be employed.Thus, in some examples, the leaving group can include —CH₂—Z,—CR¹(R²)—Z, —CH═CH—CH₂—Z, or the like, which can be used for thecontrolled release of Z. Further, while the A-Z group can be positionedat any of R³—R⁸, in some specific examples, the A-Z group can bepositioned at R⁵ or R⁸, such as in

for example. Thus, in some examples, R⁵ can include the leaving group.In other examples, R⁸ can include the leaving group.

Z can also include a variety of suitable leaving groups. Non-limitingexamples can include

(esters, carbonates, carbamates),

(Aromatic ethers),

(Phosphates and derivatives thereof),

(Hydroxamate esters),

(ammonium compounds), or the like. Thus, in some examples, the leavinggroup can include COOR¹¹, O-Aryl-R¹¹, POR¹¹R¹²R¹³⁺, ONHOR¹¹, orNR¹¹R¹²R¹³⁺, wherein R¹¹, R¹², and R¹³ are independently selected from asecond leaving group (e.g. a payload, a substrate, a reporter molecule,etc.), H, and a substituted or unsubstituted C₁-C₄ alkyl or alkylenegroup. In some specific examples, the leaving group can include COOR¹¹or POR¹¹R¹²R¹³⁺, wherein R¹¹, R¹², and R¹³ are independently selectedfrom a second leaving group (e.g. a payload, a substrate, a reportermolecule, etc.), H, and a substituted or unsubstituted C₁-C₄ alkyl oralkylene group.

In some further examples, Z can be a releasable bioactive molecule D(drug, prodrug, therapeutic agent, vitamin, cytotoxic agent, the like).The drug can be attached directly to the general structure or via animmolative or cleavable linker, as described above. Accordingly, in someexamples, the bioorthogonal molecule can have a structure such as

for example. In yet other examples, Z can be a reporter molecule (e.g.chromophore, fluorophore, profluorophore, luminophore, chemiluminophore,dye, radionuclide, or the like). In still additional examples, Z can bean affinity binder (e.g. biotin or derivatives thereof, for example).

In some examples, one or more of the substituents R¹—R⁸ or X of thebioorthogonal molecule can include a tether (T), which can be chemicallymodified or conjugated as desired. Non-limiting examples ofbioorthogonal molecules including a tether can be represented by

for example. However, the tether group need not attach at X. Asillustrated in

the tether group can be attached at any one of R¹—R⁸ or X. In somespecific examples, the tether group can be attached at one of R³—R⁸ orX. In some examples, the tether group can be attached at one of R³—R⁸.In still other examples, the tether group can be attached at X.

A tether group (T) can link the bioorthogonal molecule to a variety ofsubstrates, such as a biomolecule (e.g. glutathione, serum albumin,immunoglobulin, DNA, RNA), a homing molecule (e.g. small-moleculeligand, peptide, polypeptide), a macromolecule (e.g. polymer, dendrimer,micelle), the like, or a combination thereof.

In some examples, bioorthogonal molecules can have a structure accordingto Formula V:

which can be formed by the reaction of compositions containing abioorthogonal molecule having a structure according to Formula I andsuitable nucleophiles, in which one of thesubstituents R³—R⁸ includes areleasable group Z, one of both of R¹ and R² can include an electronwithdrawing group as described elsewhere herein, and R² can additionallyinclude an SR¹⁴ group. In some examples, R¹⁴ can include H or asubstituted or unsubstituted C₁-C₄ alkyl or alkylene group. In someexamples, R¹⁴ can be a biomolecule (e.g. glutathione, serum albumin,immunoglobulin, DNA, RNA). In some examples, a homing molecule can belinked to bioorthogonal molecule either directly or via a tether. Insome examples, R¹⁴ can be a homing molecule (e.g. small-molecule ligand,peptide, polypeptide) and the homing molecule can be linked to thebioorthogonal molecule either directly or via a tether. In someexamples, R¹⁴ can be a material or macromolecule (e.g. polymer,dendrimer, micelle), which, in some examples, can be linked to thebioorthogonal molecule either directly or via a tether. In some specificexamples, Z can be positioned at the R⁵ or R⁸ position to generate amolecule such as

or the like.

Bioorthogonal molecules according to Formula I can be prepared in avariety of ways. For example, a bioorthogonal molecule having astructure according to Formula I can be prepared by reaction with asuitable nucleophile. As one non-limiting example, a precursor moleculeof a bioorthogonal molecule having a structure according to Formula Ican include an A-OH group, such as +CH₂OH, for example, at the R³—R⁸group where it is desirable to position the A-Z group. The precursormolecule can be reacted with a suitable nucleophile to generate abioorthogonal molecule having a structure according to Formula I.Similarly, Z can include a second leaving group (e.g., 4-nitrophenyloxy,perfluorophenyloxy, succinimidyl, halide, etc., for example). Inadditional examples, Z can include a leaving group (e.g., halide,sulfonate) and that forms a bioorthogonal molecule having a structureaccording to Formula I upon reaction with a suitable nucleophile.

The bioorthogonal molecule can also be included in a therapeuticcomposition. The therapeutic composition can include an effective amountor a therapeutically effective amount of a therapeutic agent coupled tothe biorthogonal molecule in a pharmaceutically acceptable carrier. Aswill be appreciated by one skilled in the art, the effective amount ortherapeutically effective amount can be highly dependent on theparticular therapeutic agent linked to the bioorthogonal molecule.Further a variety of therapeutic agents can be linked to thebiorthogonal molecule, such as a drug, prodrug, vitamin, cytotoxicagent, other therapeutic agent, the like, or a combination thereof.Nonlimiting examples of possible therapeutic agents can includedoxorubicin, auristatins, mitomycin C, and the like.

The biorthogonal molecule can be included in a pharmaceuticallyacceptable carrier. The nature of the pharmaceutically acceptablecarrier can depend on the intended mode of administration. For example,the pharmaceutically acceptable carrier can be formulated to administerthe therapeutic composition via injection, enteral administration,topical administration, transdermal administration, transmucosaladministration, inhalation, implantation, or the like.

In some examples, the pharmaceutically acceptable carrier can beformulated to provide a therapeutic composition for administration viainjection, such as intramuscular injection, intravenous injection,subcutaneous injection, intradermal injection, intrathecal injection,intraocular injection, or the like. In such examples, thepharmaceutically acceptable carrier can include a variety of components,such as water, a solubilizing or dispersing agent, a tonicity agent, apH adjuster or buffering agent, a preservative, a chelating agent, abulking agent, the like, or a combination thereof.

In some examples, an injectable therapeutic composition can include asolubilizing or dispersing agent. Non-limiting examples of solubilizingor dispersing agents can include polyoxyethylene sorbitan monooleates,lecithin, polyoxyethylene polyoxypropylene co-polymers, propyleneglycol, glycerin, ethanol, polyethylene glycols, sorbitol,dimethylacetamide, polyethoxylated castor oils, n-lactamide,cyclodextrins, caboxymethyl cellulose, acacia, gelatin, methylcellulose, polyvinyl pyrrolidone, the like, or combinations thereof.

In some examples, an injectable therapeutic composition can include atonicity agent. Non-limiting examples of tonicity agents can includesodium chloride, potassium chloride, calcium chloride, magnesiumchloride, mannitol, sorbitol, dextrose, glycerin, propylene glycol,ethanol, trehalose, phosphate-buffered saline (PBS), Dulbecco’s PBS,Alsever’s solution, Tris-buffered saline (TBS), water, balanced saltsolutions (BSS), such as Hank’s BSS, Earle’s BSS, Grey’s BSS, Puck’sBSS, Simm’s BSS, Tyrode’s BSS, and BSS Plus, the like, or combinationsthereof. The tonicity agent can be used to provide an appropriatetonicity of the therapeutic composition. In one aspect, the tonicity ofthe therapeutic composition can be from about 250 to about 350milliosmoles/liter (mOsm/L). In another aspect, the tonicity of thetherapeutic composition can be from about 277 to about 310 mOsm/L.

In some examples, an injectable therapeutic composition can include a pHadjuster or buffering agent. Non-limiting examples of pH adjusters orbuffering agents can include a number of acids, bases, and combinationsthereof, such as hydrochloric acid, phosphoric acid, citric acid, sodiumhydroxide, potassium hydroxide, calcium hydroxide, acetate buffers,citrate buffers, tartrate buffers, phosphate buffers, triethanolamine(TRIS) buffers, the like, or combinations thereof. Typically, the pH ofthe therapeutic composition can be from about 5 to about 9, or fromabout 6 to about 8.

In some examples, an injectable therapeutic composition can include apreservative. Non-limiting examples of preservatives can includeascorbic acid, acetylcysteine, bisulfite, metabisulfite,monothioglycerol, phenol, meta-cresol, benzyl alcohol, methyl paraben,propyl paraben, butyl paraben, benzalkonium chloride, benzethoniumchloride, butylated hydroxyl toluene, myristyl gamma-picolimiumchloride, 2-phenoxyethanol, phenyl mercuric nitrate, chlorobutanol,thimerosal, tocopherols, the like, or combinations thereof.

In some examples, an injectable therapeutic composition can include achelating agent. Non-limiting examples of chelating agents can includeethylenediaminetetra acetic acid, calcium, calcium disodium,versetamide, calteridol, diethylenetriaminepenta acetic acid, the like,or combinations thereof.

In some examples, an injectable therapeutic composition can include abulking agent. Non-limiting examples of bulking agents can includesucrose, lactose, trehalose, mannitol, sorbitol, glucose, rafinose,glycine, histidine, polyvinyl pyrrolidone, the like, or combinationsthereof.

In yet other examples, the pharmaceutically acceptable carrier can beformulated to provide a therapeutic composition for enteraladministration, such as via solid oral dosage forms or liquid oraldosage forms. In the case of solid oral dosage forms, thepharmaceutically acceptable carrier can include a variety of componentssuitable for forming a capsule, tablet, or the like. In the case of aliquid dosage form, the pharmaceutically acceptable carrier can includea variety of components suitable for forming a dispersion, a suspension,a syrup, an elixir, or the like.

In some specific examples, the therapeutic composition can be formulatedas a tablet. In such examples, the therapeutic composition can typicallyinclude a binder. Non-limiting examples of binders can include lactose,calcium phosphate, sucrose, corn starch, microcrystalline cellulose,gelatin, polyethylene glycol (PEG), polyvinyl pyrrolidone (PVP),hydroxypropyl cellulose, hydroxyethylcellulose, carboxymethyl cellulose(CMC), the like, or combinations thereof.

Where the therapeutic composition is formulated as a tablet, in someexamples the therapeutic composition can also include a disintegrant.Non-limiting examples of disintegrants can include crosslinked PVP,crosslinked CMC, modified starch, sodium starch glycolate, the like, orcombinations thereof.

In some examples the tablet can also include a filler. Non-limitingexamples of fillers can include lactose, dicalcium phosphate, sucrose,microcrystalline cellulose, the like, or combinations thereof.

In some further examples, the tablet can include a coating, such as anenteric coating or other suitable coating. Such coatings can be formedwith a variety of materials, such as hydroxypropyl methylcellulose(HPMC), shellac, zein, various polysaccharides, various enterics, thelike, or combinations thereof.

In some examples, the tablet can include a variety of other ingredients,such as anti-adherents (e.g. magnesium stearate, for example),colorants, glidants (e.g. fumed silica, talc, magnesium carbonate, forexample), lubricants (e.g. talc, silica, magnesium stearate, stearicacid, for example) preservatives, desiccants, and/or other suitabletablet excipients, as desired.

In some other examples, the therapeutic composition can be formulated asa capsule. In such examples, the capsule itself can typically includegelatin, hypromellose, HPMC, CMC, the like, or combinations thereof. Avariety of excipients can also be included within the capsule, such asbinders, disintegrants, fillers, glidants, preservatives, coatings, thelike, or combinations thereof, such as those listed above with respectto tablets, for example, or other suitable variations.

In some examples, the therapeutic composition can be formulated as aliquid oral dosage form. A liquid oral dosage form can include a varietyof excipients, such as a liquid vehicle, a solubilizing agent, athickener or dispersant, a preservative, a tonicity agent, a pH adjusteror buffering agent, a sweetener, the like, or a combination thereof.Non-limiting examples of liquid vehicles can include water, ethanol,glycerol, propylene glycol, the like, or combinations thereof.Non-limiting examples of solubilizing agents can include banzalkoniumchloride, benzethonium chloride, cetylpyridinium chloride, docusatesodium, nonoxynol-9, octoxynol, polyoxyethylene polyoxypropyleneco-polymers, polyoxyl castor oils, polyoxyl hydrogenated castor oils,polyoxyl oleyl ethers, polyoxyl cetylstearyl ethers, polyoxyl stearates,polysorbates, sodium lauryl sulfate, sorbitan monolaurate, sorbitanmonooleate, sorbitan monopalmitate, sorbitan monostearate, tyloxapol,the like, or combinations thereof. Non-limiting examples of thickenersor dispersants can include sodium alginate, methylcellulose,hydroxyethylcellulose, hydroxypropylcellulose, HPMC, CMC,microcrystalline cellulose, tragacanth, xanthangum, bentonite,carrageenan, guar gum, colloidal silicon dioxide, the like, orcombinations thereof. The preservative, tonicity agent, pH adjuster orbuffering agent can typically be any of those described above withrespect to the injectable formulations or other suitable preservative,tonicity agent, pH adjuster or buffering agent. Sweeteners can includenatural and/or artificial sweeteners, such as sucrose, glucose,fructose, stevia, erythritol, xylitol, aspartame, sucralose, neotame,acesulfame potassium, saccharin, advantame, sorbitol, the like, orcombinations thereof, for example.

In yet other examples, the pharmaceutically acceptable carrier can beformulated to provide a therapeutic composition for topical,transdermal, or transmucosal administration, such as to the skin, to theeye, to the vaginal cavity, to the rectum, to the nasal cavity, thelike, or a combination thereof. Further, the topical, transdermal, ortransmucosal formulations can be formulated for local and/or systemicdelivery of one or more components of the therapeutic composition.

Where the therapeutic composition is formulated for topical,transdermal, or transmucosal administration, the pharmaceuticallyacceptable carrier can include a variety of components suitable forforming a suspension, dispersion, lotion, cream, ointment, gel, foam,patch, powder, paste, sponge, the like, or a combination thereof.Non-limiting examples can include a solubilizer, an emulsifier, adispersant, a thickener, an emollient, a pH adjuster, a tonicity agent,a preservative, an adhesive, a penetration enhancer, the like, or acombination thereof. Non-limiting examples of solubilizers and/oremulsifiers can include water, ethanol, propylene glycol, ethyleneglycol, glycerin, polyethylene glycol, banzalkonium chloride,benzethonium chloride, cetylpyridinium chloride, docusate sodium,nonoxynol-9, octoxynol, polyoxyethylene polyoxypropylene co-polymers,polyoxyl castor oils, polyoxyl hydrogenated castor oils, polyoxyl oleylethers, polyoxyl cetylstearyl ethers, polyoxyl stearates, polysorbates,sodium lauryl sulfate, sorbitan monolaurate, sorbitan monooleate,sorbitan monopalmitate, sorbitan monostearate, tyloxapol, the like, orcombinations thereof. In some examples, the solubilizer can also includea hydrocarbon or fatty substance, such as petrolatum, microcrystallinewax, paraffin wax, mineral oil, ceresi, coconut oil, bees wax, oliveoil, lanolin, peanut oil, spermaceti wax, sesame oil, almond oil,hydrogenated castor oils, cotton seed oil, soybean oil, corn oil,hydrogenated sulfated castor oils, cetyl alcohol, stearyl alcohol, oleylalcohol, lauryl alcohol, myristyl alcohol, stearic acid, oleic acid,palmitic acid, lauraic acid, ethyl oleate, isopropyl myristicate, thelike, or combinations thereof. In some examples, the solubilizer caninclude a silicon, such as polydimethylsiloxanes, methicones,dimethylpropylsiloxanes, methyl phenyl polysiloxanes, steryl esters ofdimethyl polysiloxanes, ethoxylated dimethicones, ethoxylatedmethicones, the like, or combinations thereof.

In some additional examples, the therapeutic composition can include adispersant and/or thickening agent, such as polyacrylic acids (e.g.Carbopols, for example), gelatin, pectin, tragacanth, methyl cellulose,hydroxylethylcellulose, hydroxypropylcellulose, HPMC, CMC, alginate,starch, polyvinyl alcohol, polyvinyl pyrrolidone, co-polymers ofpolyoxyethylene and polyoxypropylene, polyethylene glycol, the like, orcombinations thereof.

In some examples, the therapeutic composition can include an emollient,such as aloe vera, lanolin, urea, petrolatum, shea butter, cocoa butter,mineral oil, paraffin, beeswax, squalene, jojoba oil, coconut oil,sesame oil, almond oil, cetyl alcohol, stearyl alcohol, olive oil, oleicacid, triethylhexanoin, glycerol, sorbitol, propylene glycol,cyclomethicone, dimethicone, the like, or combinations thereof.

In some examples, the therapeutic composition can include an adhesive,such as acrylic adhesives, polyisobutylene adhesives, silicon adhesives,hydrogel adhesives, the like, or combinations thereof.

In some examples, the therapeutic composition can include a penetrationenhancer, such as ethanol, propylene glycol, oleic acid and other fattyacids, azone, terpenes, terpenoids, bile acids, isopropyl myristate andother fatty esters, dimethyl sulphoxides, N-methyl-2-pyrrolidone andother pyrrolidones, the like, or combinations thereof.

The pH adjusters, tonicity agents, and preservatives in the topical,transdermal, or transmucosal therapeutic composition can generallyinclude those pH adjusters and buffering agents, tonicity agents, andpreservative agents listed above, or any other suitable pH adjusters,buffering agent, tonicity agent, or preservative for a particularformulation and/or use thereof. In some examples, the therapeuticcomposition can also include fumed silica, mica, talc, titanium dioxide,kaolin, aluminum glycinate, ethylenediaminetetraacetic acid, fragrances,colorants, other components as described above, the like, orcombinations thereof.

In some additional examples, the pharmaceutically acceptable carrier canbe formulated for administration via inhalation. In some examples, suchformulations can include a propellant, such as hydrofluoralkanes, suchas HFA134a, HFA227, or other suitable propellant. In yet other examples,the therapeutic composition can be formulated for administration vianebulization. In either case, the therapeutic composition can alsoinclude a variety of solubilizing agents, such as those described above.In other examples, the therapeutic composition can be formulated as adry powder aerosol. In some examples, the therapeutic composition caninclude a particulate carrier and/or other particulate excipients, suchas lactose, mannitol, other crystalline sugars, fumed silica, magnesiumstearate, amino acids, the like, or combinations thereof.

In some specific examples, the pharmaceutically acceptable carrier canbe formulated to provide a therapeutic composition for ocularadministration. Non-limiting examples can include topical application tothe eye in the form of a drop, a gel, a film, an insert, a sponge, anointment, the like, or a combination thereof. In yet other examples, thetherapeutic composition can be formulated for intraocular injection orimplantation in the form of a solution, a depot, a scaffold, the like,or a combination thereof. Ocular compositions can include a variety ofexcipients, such as water, a tonicity agent, a thickening agent, abiodegradable polymer, a solubilizing agent, an emulsifier, apreservative, the like, or other suitable component, or a combinationthereof. In some examples, the ocular composition can include abiodegradable polymeric matrix that can include a variety ofbiodegradable constituents, such as polylactic acid,poly(lactic-co-glycolic) acid, polyglycolic acid, poly(caprolactone),hyaluronic acid, polyhydroxybutyrate, polyvinyl alcohol,polyvinylpyrrolidone, carbomers, polyacrylic acid,polyoxyethylene/polyoxypropylene copolymers, other copolymers, albumins,casein, zein, collagen, other proteins, glucose, sucrose, maltose,trehalose, amylose, dextrose, fructose, mannose, galactose, othersugars, erythritol, threitol, arabitol, xylitol, ribitol, mannitol,sorbitol, galactitol, fucitol, iditol, inositol, volemitol, isomalt,maltitol, lactitol, maltotriitol, maltotetraitol, polyglycitol, othersugar alcohols, chondroitin and/or other glycosaminoglycans, inulin,starches, acacia gum, agar, carboxymethyl cellulose, methyl cellulose,ethyl cellulose, alginates, carrageenan, cassia gums, cellulose gums,chitin, chitosan, curdlan, gelatin, dextran, fibrin, fulcelleran, gellangum, ghatti gum, guar gum, tragacanth, karaya gum, locust bean gum,pectin, starch, tara gum, xanthan gum, and other polysaccharides, andfunctionalized derivatives of any of the above, copolymers thereof, thelike, or mixtures thereof.

It is noted that a variety of components are listed for use in specificcarriers or carrier types. However, the lists of components are notnecessarily intended to be exclusive to a particular carrier or carriertype. For example, the biodegradable polymers specifically listed withreference to ocular formulations may also be useful in other carriers orcarrier types as well. Thus, where suitable, any of the componentsdisclosed herein can be employed in any pharmaceutically acceptablecarrier whether or not the particular component is specifically listedwith specific reference to a particular carrier type.

In some examples, the therapeutic compositions described herein can bedisposed in a suitable container. Such containers can includemultiple-use containers or single use containers. Non-limiting examplescan include bottles, vials, blister packs, bags, or the like. In someexamples, the container can be an amber colored container or othersuitable container configured to protect the dosage form or therapeuticcomposition from light. In yet other examples, the container can includeinstructions and dosing information for the dosage form or therapeuticcomposition. The container can include a variety of materials, such aspolyethylene, polypropylene, polycarbonate, polyvinyl chloride, glass,the like, or a combination thereof.

Generally, the leaving group Z of the bioorthogonal molecule will remaincovalently attached to the bioorthogonal molecule until thebioorthogonal molecule interacts with a releasing molecule, such as atetrazine molecule. In some examples, the releasing molecule can have astructure according to Formula III or Formula IV:

or

With this in mind, in some examples, the therapeutic composition caninclude a releasing molecule, such as a tetrazine molecule, to reactwith the bioorthogonal molecule to release leaving group Z. Thus, insome examples, the bioorthogonal molecule and the releasing molecule canbe included in the same composition and in the same container. Wherethis is the case, it may be desirable to minimize interaction betweenthe bioorthogonal molecule and the releasing molecule prior toadministration by adjusting the pH to minimize reactivity between thebioorthogonal molecule and the releasing molecule prior toadministration, reducing solubility of the bioorthogonal molecule and/orthe releasing molecule in the composition to minimize reactivity betweenthe bioorthogonal molecule and the releasing molecule prior toadministration, increasing the viscosity of the composition to minimizereactivity between the bioorthogonal molecule and the releasing moleculeprior to administration, reducing the temperature of the composition tominimize reactivity between the bioorthogonal molecule and the releasingmolecule prior to administration, the like, or a combination thereof. Insome examples, mere administration can provide a suitable reactionenvironment for the bioorthogonal molecule and the releasing molecule.In other examples, adjustments can be made to the composition prior toadministration to provide a suitable administration vehicle and/orreaction environment.

The present disclosure also describes a therapeutic system. Thetherapeutic system can include a therapeutic composition and a separatereleasing composition. The therapeutic composition can include abioorthogonal molecule as described herein and a pharmaceuticallyacceptable carrier, also as described herein. The releasing compositioncan include a releasing molecule and a second pharmaceuticallyacceptable carrier, as described herein.

In some examples, the therapeutic composition and the releasingcomposition can be disposed in separate containers. In some cases, thiscan prevent premature release of the therapeutic agent from thebioorthogonal molecule. In some examples, the therapeutic compositionand the releasing composition can be mixed immediately prior toadministration (e.g. within about 5 minutes, 10 minutes, 30 minutes, or60 minutes). In other examples, the therapeutic composition and thereleasing composition can be administered separately so that theindividual compositions are not mixed prior to administration. It isnoted that in some examples, the therapeutic composition and thereleasing composition can have the same pharmaceutically acceptablecarrier. In other examples, the therapeutic composition and thereleasing composition can have distinct pharmaceutically acceptablecarriers.

In some examples, the therapeutic composition and the releasingcomposition can be included in the same container. Where this the case,the common container may include a rupturable or otherwise removablemembrane or barrier separating the therapeutic composition and thereleasing composition. In some examples, the therapeutic composition andthe releasing composition can be disposed in a syringe having acork-screw mixer or the like to mix the therapeutic composition and thereleasing composition as they are dispensed from the syringe. Othersuitable arrangements of the therapeutic composition and the releasingcomposition in the common container can also be employed.

A variety of releasing molecules can be used in the releasingcomposition. Generally, any suitable tetrazine-based molecule can beused or other suitable compound for releasing the payload from thebioorthogonal molecule. In some specific examples, the releasingmolecule can have a structure according to Formula III:

In some additional examples, the releasing molecule can have a structureaccording to Formula IV:

The therapeutic compositions or therapeutic systems can be employed in avariety of treatment regimens or dosing regimens. For example, in somecases, the therapeutically effective amount can be administered via asingle dose and/or a dosage regimen. In some examples, the dosageregimen can include administering the therapeutic composition at asuitable frequency. In some examples, the dosage regimen can includeadministering the therapeutic composition from 1 time per day to 12times per day or more in individual doses. In some further examples, thedosage regimen can include administering the therapeutic compositionfrom 1 time per day to 2, 3, 4, or 6 times per day in individual doses.In yet other examples, the therapeutic composition can be administeredvia infusion, or other equivalent process. Where this is the case, insome examples, the composition can be administered over a period of fromabout 30 minutes or 1 hour to about 6 hours or 12 hours or more.Further, depending on the adverse health condition, administration ofthe therapeutic composition can be performed over a period of from 1 dayto 365 days or more, over a period from 1 day to 30 days, over a periodof 7 days to 90 days, over a period of 1 month to 6 months, 12 months,18 months, or 24 months, or other suitable treatment period at anysuitable frequency, such as those described above, or other suitablefrequency, such as once per week, twice per week, three times per week,once every two weeks, once per month, once every six weeks, once everytwo months, etc.

The bioorthogonal molecules described herein can have a variety of usesand can be employed in a variety of methods. For example, in some cases,the bioorthogonal molecule can be used as a protecting group that can beremoved under mild conditions, including in biological samples and invivo. This can be done in a variety of ways. For example, the structureof a target molecule can be temporarily modified with a reactiveprecursor of the bioorthogonal molecule, followed by subsequent removalof the bioorthogonal molecule from the target molecule by contact with areleasing molecule. In another example, the bioorthogonal molecule canbe incorporated onto a target molecule during its synthesis, followed bysubsequent removal of the bioorthogonal molecule from the targetmolecule by contact with a releasing molecule

In some examples, a target molecule can be inactivated by derivatizationwith the bioorthogonal molecule, such that upon reaction with suitablemolecules, such as a releasing molecule, the target molecule isreactivated by loss of the bioorthogonal molecule. In some examples, thebioorthogonal molecule can be attached to the modified target moleculevia an immolative tether.

In some examples, polypeptides (e.g. proteins) or derivatives thereofcan be modified at one or several of: amino-acid side chains (e.g. —NH₂of Lys; —OH of Ser, Thr, Tyr; —SH of Cys), the amino- orcarboxy-terminus, at modified side chains (e.g. phosphorylated Ser, Thr,Tyr), or at artificial amino acids part of the polypeptide sequence withthe bioorthogonal molecule (e.g. BNBD), as illustrated in FIG. 1A.Specifically, FIG. 1A illustrates a target molecule 104 that has beenreversibly modified with a bioorthogonal molecule (e.g. BNBD). Whilespecific reference will be made to polypeptides with respect to FIGS.1A-1C, the modified target molecule 104 can represent a variety oftarget molecules, such as a polypeptide, a carbohydrate, a lipid, anucleic acid, or the like. The bioorthogonal molecule can be releasedfrom the modified target molecule 104 (e.g. a polypeptide) byinteraction with a releasing group (e.g. tetrazine, for example).

Such modified target molecules can be prepared in a variety of ways. Forexample, as illustrated in FIG. 1B, a bioorthogonal molecule precursor(BNBD-Z), having a suitable leaving group, can be reacted with asuitable target molecule 102. This can removably couple thebioorthogonal molecule (e.g. BNBD) to the target molecule 102 to providea modified target molecule 104. Suitable modified polypeptides and othersuitable target molecules can also be prepared by expanded-genetic codemethods or by adding the bioorthogonal molecule to the target moleculeduring the synthesis of the target molecule.

In some additional examples, modified polypeptides can be prepared bylinking two precursor polypeptides (e.g. native chemical ligation,intein ligation), at least one of which is modified with thebioorothogonal molecule. The bioorthogonal molecules can also be used toperform enzymatic labeling substrates containing the bioorthogonalmolecule.

In some examples, a polypeptide can be reconstituted by contact withsuitable bioorthogonal molecules (e.g. BNBD), followed by removal byreaction with a suitable releasing group (e.g. Tz) as illustrated inFIG. 1C. In this example, modified target molecule 104 can be reactedwith a releasing group to produce a reconstituted polypeptide 106.

In still additional examples, the activity of an enzyme can becontrollably reconstituted by contacting and subsequently dissociating asuitable bioorthogonal molecule from the enzyme using a releasing group,as illustrated in FIG. 2 . Specifically, the enzyme 104 can be modifiedby removably coupling a bioorthogonal molecule at any position thatinactivates its function. The bioorthogonal molecule can be subsequentlyreleased from the inactive enzyme 104 using the releasing group toproduce an active enzyme 106 having reconstituted activity.

In some further examples, the controlled reconstitution of enzymeactivity can be associated with the generation of an analytic signal(e.g. fluorescence, bioluminescence, chemiluminescence, colorimetric,etc.). In additional examples, the controlled reconstitution of enzymeactivity can be associated with the generation of a therapeutic agent.In still additional examples, the controlled reconstitution of enzymeactivity can be associated with the activation of cytotoxic enzymeactivity (e.g. enzyme toxins).

In some examples, a bioorthogonal molecule can be used to spatiallyand/or temporally control the activation of an enzyme. For example, amodified enzyme (i.e. modified with a bioorthogonal molecule) can bedelivered to a specific location (e.g. tissue, tumor), followed byrelease of the bioorthogonal molecule via a releasing molecule andassociated activation of the enzyme. In some further examples, enzymeactivation can be followed by subsequent activation of prodrugs,activatable reporter groups, or can be associated with a therapeutic orcytotoxic effect.

In still additional examples, the bioorthogonal molecules can be used inmethods for the controlled release of a polypeptide for interactionswith other biomolecules (e.g. proteins, nucleic acids, etc.), asillustrated in FIG. 3 , for example. Specifically, polypeptide 104 canbe modified by removably coupling a bioorthogonal molecule (e.g. BNBD)thereto to prevent interaction with biomolecule 108. When interactionwith biomolecule 108 is desired, the bioorthogonal molecule can beremoved via reaction with a releasing group to prepare reconstitutedpolypeptide 106 having restored interaction with biomolecule 108.

Bioorthogonal molecules can also be used in methods for controlled drugdelivery. For example, a modified polypeptide can be delivered to aspecific location (e.g. tissue, tumor) followed by activation of thepolypeptide by release of the bioorthogonal molecule with a suitablereleasing molecule. In some examples, the activated protein can be acytokine, chemokine, enzyme hormone, protein toxin, the like, or acombination thereof.

In additional examples, bioorthogonal molecules can be used in methodsfor the controlled release of a polypetide therapeutic (e.g. cytokine,chemokine, peptide hormone, protein toxin). In yet other examples,bioorthogonal molecules can be used in methods for the controlledrelease of a polypeptide (e.g. receptor) for interacting with smallmolecules, as illustrated in FIG. 4 . Specifically, a small-moleculebinding pocket of modified polypeptide 104 can be blocked to removablycoupling a bioorthogonal molecule at or near the binding pocket. Thiscan prevent interaction of the protein ligand 108 with the polypeptide.When interaction is desired, a removing molecule can be introduced toremove the bioorthogonal molecule to produce a reconstituted polypeptide106 having restored protein ligand 108 binding.

In some examples, molecules including oligonucleotides (e.g. DNA, RNA)or derivatives thereof (e.g. PNA, LNA, 2′-OMe-RNA, phosphorothioates)can be modified at nucleobases, the termini, and/or the backbone withone or more bioorthogonal molecules (e.g. BNBD). For example,bioorthogonal molecules can be directly attached to the specifiedresidues or via immolative spacers. As illustrated in FIG. 5A, nucleicacid 110 can have bioorthogonal molecules attached at select nucleobases112, on the backbone 114, or both, as desired. The following structuresrepresent some examples of attachment points for the bioorthogonalmolecule to a nucleic acid. For example, in some cases, thebioorthogonal molecule can be attached directly to the specificnucleobase as follows:

for example. In other examples, the bioorthogonal molecule can beattached to the nucleic acid at the backbone, such as in the followingstructure:

Regardless of the particular attachment point for the bioorthogonalmolecules to the nucleic acid, the bioorthogonal molecules can be usedin a variety of methods employing nucleic acids. For example, in somecases, the bioorthogonal molecules can be used in methods toreconstitute the structure of an oligonucleotide or a polynucleotide bycontacting the bioorthogonal molecule with a releasing molecule torelease the underlying oligonucleotide. One such example is illustratedin FIG. 5B. Specifically, a variety of bioorthogonal molecules areattached to a nucleic acid (e.g. an oligonucleotide or a polynucleotide)to form a modified nucleic acid 110. As desired, the bioorthogonalmolecule can be reacted with a releasing group to produce areconstituted nucleic acid 116.

The bioorthogonal molecules can also be used in methods to control thehybridization of oligonucleotides or polynucleotides via removal ofbioorthogonal molecule modifications using a releasing molecule torelease the target nucleic acid. One specific example of this isillustrated in FIG. 5C. Specifically, a target nucleic acid 111 can beprevented from hybridizing with modified nucleic acid probe 110 due tothe removable coupling of a bioorthogonal molecule to the nucleic acidprobe 110. Reaction with a releasing group can remove the bioorthogonalmolecule from nucleic acid probe 110 to allow hybridization and toprepare a reconstituted nucleic acid 118.

Similarly, the bioorthogonal molecules can also be used in methods tocontrol the folding of oligonucleotides or polypeptides by removal ofbioorthogonal molecule modifications by reaction with a releasingmolecule. For example, as illustrated in FIG. 5D, modified nucleic acid110 can be prevented from folding due to the presence of bioorthogonalmolecules removably coupled thereto. Reaction with a releasing group canremove the bioorthogonal molecules from the nucleic acid 110 to allowproper folding of the nucleic acid and preparation of a reconstitutednucleic acid 118. In some examples, the nucleic acid (e.g. anoligonucleotide) can be an aptamer or ribozyme.

In some examples, the bioorothogonal molecules can be used in methodsfor the synthesis of BNBD-modified oligonucleotides by reactingphosphorothioates with bioorthogonal molecules having a suitable leavinggroup (e.g. BNBD-Z, where Z is suitable leaving group.) On example ofsuch a reaction is illustrated in FIG. 5E. In some other examples, thebioothogonal molecules can be used in methods for the synthesis ofmodified oligonucleotides by incorporation of modified nucleotidederivatives during oligonucleotide solid phase synthesis. In someexamples, the bioorthogonal molecules can be precursors for thesolid-phase synthesis of modified oligonucleotides (for example byphosphite or phosphoramidite method). In some examples, the precursorscan also be modified on the nucleobase. One non-limiting examples isdepicted below for illustrative purposes only.

Additionally, the bioorthogonal molecules can also be used in methodsthat remove bioorthogonal molecule modifications from oligonucleotidebackbones by reaction with a releasing molecule. In some examples, thebackbone can have a phosphate or phosphorothioate structure. One exampleof such a reaction is illustrated in FIG. 5F. In some specific examples,the bioorthogonal molecules can be used in methods for the removal of abioorthogonal molecule from an oligonucleotide terminus by reaction witha suitable releasing molecule. In some examples, a method can includemultiple cycles of incorporating a nucleotide containing a modificationof the bioorthogonal molecule and removal of the bioorthogonal moleculeby contact with a suitable releasing molecule.

In some examples, the bioorthogonal molecules can be used in methodsthat control the dissociation of oligonucleotides by removal of one ormore bioorthogonal molecule modifications from the backbone, asillustrated in FIG. 5G, for example. Specifically, modified nucleic acid120 can include bioorthogonal molecule modifications that stabilize themodified nucleic acid 120 or otherwise prevent the dissociation of theindividual nucleic acid strands. Reaction with a releasing group canremove the bioorthogonal molecule modifications from the modifiednucleic acid 120 to prepare a nucleic acid 122 that is less stable orotherwise prone to dissociation. The nucleic acid 122 can dissociateinto separate nucleic acid strands 124A, 124B.

In some additional examples, the bioorthogonal molecules can be used inmethods for cell delivery of oligonucleotides and intracellularactivation of oligonucleotides. The methods can include modifying anoligonucleotide with the bioorthogonal molecule to make it permeable toa membrane that is otherwise impermeable to the free oligonucleotide(e.g. cell membrane) followed by removal of the modifications by contactwith a releasing molecule, causing the retention of the oligonucleotidewithin the compartment formed by the impermeable membrane (e.g. cell).

In some examples, the bioorthogonal molecules can be used in methods toelucidate the composition of an oligonucleotide molecule (e.g. DNAsequencing) by sequential incorporation of one or several nucleotidesresulting in the modification of one of the termini (such as the 3′terminus, for example) with a bioorthogonal molecule, a reading step,and removal of the modification.

In some examples, the bioorthogonal molecule can include a precursor forthe enzymatic incorporation of modified nucleotides into anoligonucleotide sequence. The bioorthogonal molecule modification can belinked to the oligonucleotide directly or via a immolative linker. Insome examples, the precursor molecules can be modified at any positionof the nucleobase or carbohydrate, for example.

In some examples, the bioorthogonal molecule (sometimes referred toherein as BNBD) can have a structure that includes a targeting moiety(abbreviated L), a tether (abbreviated as T), and a leaving group(abbreviated as Z), such as L-T-BNBD-Z or L-T-Z-BNBD, for example. Thetargeting moiety can be but is not restricted to small-molecules,polypeptides, oligonucleotides, polymers, liposomes, micelles, or thelike. In some examples, the releasing molecule (abbreviated Tz) caninclude a targeting moiety (abbreviated L) and a tether (abbreviated asT), such as L-T-Tz, for example. In some examples, compositions caninclude a bioorthogonal molecule and a releasing molecule, in which thetargeting moieties L and L′ bind to proximal sites of a common target.For example, FIG. 6A illustrates a bioorthogonal molecule L-T-BNBD-Zbound to a first site of a target molecule 130 and a releasing moleculeL′-T′-Tz bound to a site proximal to the first site. FIG. 7A illustratesanother example of a bioorthogonal molecule L-T-Z-BNBD bound to a firstsite of a target molecule 130 and a releasing molecule L′-T′-Tz bound toa site proximal to the first site.

It is noted that the various combinations of targeting moieties andtarget molecules are not particularly limited, and that any suitabletargeting moiety, target molecule, or combinations thereof can beemployed. In some specific examples, L and L′ can be small molecules andthe target molecule can be a polypeptide (including multimeric proteinsand protein complexes). In additional examples, L and L′ can beoligonucleotides (e.g. aptamers) and the target molecule can be apolypeptide (including multimeric proteins and protein complexes). Instill additional examples, L and L′ can be polypeptides and the targetmolecule can be a polypeptide (including multimeric proteins and proteincomplexes). In some specific examples, L and L′ can be antibodies orfragments thereof.

Similarly, the leaving group Z is also not particularly limited and anysuitable leaving group can be employed. In some specific examples, Z canbe a reporter group (e.g. fluorophore, chemiluminophore, bioluminophore,radionuclide), affinity binder (e.g. hapten, biotin), a therapeuticagent, or the like.

In some examples, compositions can include both a bioorthogonal moleculeand a releasing molecule in which L and L′ are oligonucleotides and thetarget is a nucleic acid. For example, as illustrated in FIGS. 6B and7B, both the biorthogonal molecule and the releasing molecule caninclude a oligonucleotide targeting moieties that can hybridize to orotherwise bind to a common target nucleic acid molecule.

In some examples, as illustrated below, compositions can include abioorthogonal molecule (abbreviated as BNBD-Z or Z-BNBD), a tether(abbreviated as T), a releasing group (abbreviated as Tz), and a moietywith affinity to each other (abbreviated as C):

or the like. The interaction between C and C′ can be any suitableinteraction. In some specific examples, the interaction between C and C′can be a covalent interaction. In other examples, the interactionbetween C and C′ can be a non-covalent interaction.

In some examples, C and C′ can be oligonucleotides. Some non-limitingexamples of such structures are illustrated in FIGS. 6C and 7C. In someexamples, where C and C′ are oligonucleotides, the oligonucleotides canbe modified with targeting molecules (abbreviated as L) and can furtherinclude tethers (abbreviated T). Non-limiting examples of suchstructures are illustrated in FIGS. 6D and 7D. However, it is noted thatthe various elements of the bioorthogonal molecule and releasingmolecule can be arranged in any suitable configuration.

In some additional examples, the targeting moieties can bind to proximalsites on an analyte molecule or target molecule (e.g.biomacromolecules). For example, FIGS. 6E and 7E each illustratedifferent arrangements of bioorthogonal molecules bound to proximalsites on a target molecule 130.

In some specific examples, L and L′ can be small molecules and thetarget molecule can be a polypeptide (including multimeric proteins andprotein complexes). In some examples, L and L′ can be oligonucleotides(e.g. aptamers) and the target molecule can be a polypeptide (includingmultimeric proteins and protein complexes). In yet additional examples,L and L′ can be polypeptides and the target molecule can be apolypeptide (including multimeric proteins and protein complexes). Instill additional examples, L and L′ can be antibodies or fragmentsthereof.

In some additional examples, Z can be a reporter group (e.g.fluorophore, chemiluminophore, bioluminophore, radionuclide), affinitybinder (e.g. hapten, biotin), therapeutic agent, or the like.

The bioorthogonal molecules can also be used in methods for thedetection of an analyte (such as a biomacromolecule, for example) wherea reporter signal can be amplified by repeated contacting of thebioorthogonal molecules with the target molecule, proximity-acceleratedreaction of the molecules, release of Z, and dissociation of themolecules.

In some examples, the bioorthogonal molecules can be used in methods forthe detection of an analyte (such as a biomacromolecule, for example)where the release of Z is linked to a reporter signal (e.g. fluorescenceturn-on, activation of MRI contrast agent, chemiluminescence signal,bioluminescence signal). In some examples, the bioorthogonal moleculeand target molecule can include a quencher/fluorophore pair.

In some examples, the bioorthogonal molecule can be used in methods forthe detection of an analyte (such as a biomacromolecule, for example)where the reporter signal is amplified by repeated contacting of thebioorthogonal molecule with the target molecule, proximity-acceleratedreaction of the molecules, release of Z, and dissociation of themolecules. One example of this is illustrated in FIG. 6F. Specifically,bioorthogonal molecule (L-T-BNBD-Z) and releasing molecule (L′-T′-Tz)can bind to target analyte 130 at proximal binding sites. This canfacilitate proximity-accelerated reaction of the bioorthogonal moleculeand the releasing molecule, resulting in the release of Z (reportedmolecule) and the subsequent dissociation of the side products (L-T-Iand L′-T′-P) from the target analyte 130.

In some examples, the target molecule can be a biomarker. In someexamples, the bioorthogonal molecules can be used in methods fordelivering or localizing a therapeutic agent or reporter molecule inwhich a homing molecule that binds to a biomarker is modified with atemplate molecule that can be targeted by compositions as describedherein. In some examples, the template molecule can be anoligonucleotide.

In some examples, the bioorthogonal molecules can be used in methods fordelivering or localizing a therapeutic agent or reporter molecule inwhich proximal binding of two homing molecules reveal a templatemolecule that can be targeted by compositions as described herein.

In some examples, the bioorthogonal molecules can be used in methods ofspatiotemporally controlled release of therapeutics or imaging agents inwhich compositions described herein are co-administered simultaneouslyor sequentially with a time-delay by any means of administration (e.g.topical, orally, intravenously, intramuscularly).

In some examples, the bioorthogonal molecules can be used in methods ofspatiotemporally controlled release of therapeutics or imaging agents inwhich compositions described herein can accumulate at specific locationssuch as but not restricted to a specific tissue (e.g. tumor) or organ(e.g. bladder, kidney, liver).

In some examples, the bioorthogonal molecules can be used in methods ofdelivering molecules into a cell or other objects (e.g. organelle,epidermis) with a partially permeable membrane, in which the molecule ofinterest is modified with moieties of the bioorthogonal molecule to bepermeable to the membrane (e.g. plasma membrane). In a subsequent step,contact with releasing molecules can remove the bioorthogonal molecules,which can cause retention of the molecules of interest within theenclosed space of the membrane (i.e. cell) because of reduced membranepermeability.

In some examples, compositions can include the bioorthogonal moleculeand/or the releasing molecule in which the molecules are modified withmoieties that lead to preferred accumulation at specific locations suchas but not restricted to a specific tissue (e.g. tumor) or organ (e.g.bladder, kidney, liver).

In some examples, compositions can include the bioorthogonal moleculeand/or the releasing molecule in which the molecules are modified withmoieties that improve the pharmacokinetic properties and reaction withthe releasing molecule as a method for slow release of the therapeuticagent Z.

In some examples, compositions can include the bioorthogonal moleculeand/or the releasing molecule in which Z is a therapeutic agent that canbe unstable to storage and handling but whose stability is enhanced bymodification with a bioorthogonal molecule.

In some examples, compositions of molecules can include thebioorthogonal molecule and/or the releasing molecule in which Z is atherapeutic agent that can be unstable throughout the trajectory ofadministration (e.g. in digestive system), but whose stability isenhanced by modification with a bioorthogonal molecule. In someexamples, Z can be a molecule with poor oral bioavailablility.

In some examples, target molecules can include a carrier molecule (e.g.protein, oligonucleotide, colloid, nanoparticle, liposome, micelle,dendrimer, surface, polymer, viral particle, cell surface, hydrogel,small molecule) modified with one or more bioorthogonal molecules(abbreviated BNBD-Z) conjugated either directly or via a tether(abbreviated T). In some examples, the carrier molecule leads toaccumulation at a specific anatomical localization (e.g. tissue, organ)and/or endows beneficial pharmacokinetic properties. Multiplebioorthogonal molecules with the same or different leaving groups Z canbe attached to one or more carrier molecules. In one example, two ormore different therapeutic agents can be attached to a single carriermolecule. In another example, the target molecule (via multiplebioorthogonal molecules) can include both releasable therapeutic agentsand releasable reporter molecules. Non-limiting examples of this areillustrated in FIGS. 8A and 9A, which depict the target molecule 140including a carrier molecule 142 and a plurality of bioorthogonalmolecules bound to the carrier molecule 142 via a tether group.

In some examples, compositions can include a releasing molecule(abbreviated as Tz) and a carrier molecule modified with one or morebioorthogonal molecules (shown as BNBD-Z), in which the releasingmolecule triggers the release of Z. For example, as illustrated in FIGS.8B and 9B, a target molecule 140 or 150 can include a carrier molecule142 or 152 having a plurality of bioorthogonal molecules attachedthereto via a tether T. Reaction with a releasing group can release Zfrom the bioorthogonal molecule. In some examples, as illustrated inFIG. 8B, Z can be released into solution. In other examples, asillustrated in FIG. 9B, Z can remain attached in a liberated form on thecarrier molecule 152.

In some examples, compositions can include a first carrier moleculemodified with one or more releasing molecules (shown as Tz), and asecond carrier molecule modified with one or more bioorthogonalmolecules. In this example, the first and second carrier molecules caninteract to trigger the release of Z. Non-limiting examples areillustrated in FIGS. 8C and 9C. Specifically, a releasing molecule 146or 156 can include a first carrier molecule 141 or 151 having aplurality of releasing groups attached thereto. A first target moleculecan include a second carrier molecule 142 or 152 including a pluralityof bioorthogonal molecules attached thereto. Interaction between thereleasing molecule 146 or 156 and the target molecule 140 or 150 canproduce a first product molecule 148 or 158 and a second productmolecule 144 or 154. In some examples, as illustrated in FIG. 8C, Z canbe released into solution. In other examples, as illustrated in FIG. 9C,Z can remain attached in a liberated form on the carrier molecule 152.

In some examples, compositions can include a bioorthogonal molecule(abbreviated as BNBD-Z) and a carrier molecule modified with one or morereleasing molecules (shown as Tz ). Reaction between the bioorthogonalmolecule and the releasing molecule can trigger the release of Z. Onenon-limiting example is illustrated in FIG. 10A.

In some examples, the bioorthogonal molecules can be used in methods ofspatiotemporally controlled release of therapeutics or imaging agents inwhich compositions including a bioorthogonal molecule or a releasingmolecule, at least one of which is linked to a carrier molecule, areco-administered simultaneously or sequentially with a time delay by anymeans of administration (e.g. topical, orally, intravenously,intramuscularly).

In some examples, the biooorthogonal molecules can be used in methods ofspatiotemporally controlled release of therapeutics or imaging agents inwhich carrier molecules with attached bioorthogonal molecules areimplanted at a specific location (e.g. hydrogel, stint, biomaterial) andadministration of releasing molecules releases Z.

In some examples, the bioorthogonal molecules can be used in methods ofspatiotemporally controlled release of therapeutics or imaging agents inwhich carrier molecules with attached releasing molecules are implantedat a specific location (e.g. hydrogel, stint, biomaterial) andadministration of bioorthogonal molecules releases Z.

In some examples, bioorthogonal molecules can have a structureX-T-BNBD-Z, in which BNBD stands for a bioorthogonal molecule having ageneral structure according to Formula I, and where X is a group thatreacts with cycloalkenes (e.g. cis-cyclootene, cyclorpropene), Z is aleaving group, and T is a tether. In some examples, the bioorthogonalmolecule can be modified with additional functional groups or reportermolecules (e.g. fluorophores, radionuclides, haptens, affinity binders).

In some examples, bioorthogonal molecules can have a structureY-T-BNBD-Z, in which BNBD stands for molecules having a generalstructure according to Formula I, Y is a group that is inert tocycloalkenes (e.g. cis-cyclootene, cyclorpropene, norbornene) butorthogonally reacts with other groups (example of reagent pairs:azide/cyclooctyne), Z is a releasable group, and T is a tether. In someexamples, the bioorthogonal molecules can be modified with additionalfunctional groups or reporter molecules (e.g. fluorophores,radionuclides, haptens, affinity binders).

In some examples, releasing molecules containing two or more releasingmolecules or groups (abbreviated Tz) can be attached to a carrier ormodified with a reporter or other molecule (T is tether, wavy lineindicates site of modification or attachment, B indicates branch point),such as:

or the like.

In some examples, releasing molecules or groups can be attached tocarrier molecules or reporter molecules (abbreviated R) including forexample fluorophores, radionuclides, haptens, chromophores, or the like.Non-limiting examples can include:

or the like.

In some examples, molecules can include one or more bioorthogonalmolecules (abbreviated as BNBD-Z) and a group that reacts with releasingmolecules and are attached to a carrier or modified with a reporter orother molecule (abbreviated X, T is a tether, wavy line indicates siteof modification or attachment, B indicates branch point).

In some examples, molecules can include one or more bioorthogonalmolecules of (abbreviated as BNBD-Z) and a group (abbreviated Y) that isunreactive to releasing molecules but orthogonally reacts with othergroups (e.g. azide, cyclooctyne, etc.) and are attached to a carrier ormodified with a reporter or other molecule (abbreviated X, T is atether, wavy line indicates site of modification or attachment, Bindicates branch point).

In some specific examples, these molecules can be attached to carriermolecules or reporter molecules (abbreviated R) including for examplefluorophores, radionuclides, haptens, chromophore. Non-limiting examplescan include:

or the like.

In some examples, compositions including a molecule having a structureX-T-BNBD-Z and a carrier molecule (e.g. protein, oligonucleotide,colloid, nanoparticle, liposome, micelle, dendrimer, surface, polymer,viral particle, cell surface, hydrogel) can be modified with two or morereleasing molecules (shown as Tz), which can lead to covalent linkage ofthe former molecules to the releasing molecule with simultaneous releaseof Z. (X′: linker structure formed by the reaction of Tz and X; P and I:side-products formed by the reaction of BNBD and Tz). Non-limitingexamples of such reactions are illustrated in FIGS. 10B and 10C. In someexamples, as illustrated in FIG. 10B, the released group Z can beliberated into solution. In other examples, as illustrated in FIG. 10C,Z can remain attached to the carrier molecule and side product I can bereleased into solution.

In other examples, a carrier molecule (e.g. protein, oligonucleotide,colloid, nanoparticle, liposome, micelle, dendrimer, surface, polymer,viral particle, cell surface, hydrogel) can be modified with two or moremolecules or groups (W) that form a covalent linkage with Y (Y′) andreleasing molecules (Tz) that release Z. (P and I: side-products formedby the reaction of BNBD and Tz). In some examples, as illustrated inFIG. 11A, the released moiety Z can be liberated into solution. In otherexamples, as illustrated in FIG. 11B, Z can remain attached to thecarrier molecule and side product I can be released into solution.

In some examples, compositions can include a first molecule thatincludes two or more releasing molecules (abbreviated as Tz-Tz) and asecond molecule including a carrier molecule (e.g. protein,oligonucleotide, colloid, nanoparticle, liposome, micelle, dendrimer,surface, polymer, viral particle, cell surface, hydrogel) modified withtwo or more biorthogonal molecules (shown as BNBD-Z) as well as a secondtype of molecule that reacts with releasing molecules (shown as X),which lead to covalent linkage of the former molecules to the lattermolecule with simultaneous release of Z. (X′: linker structure formed bythe reaction of Tz and X; P and I: side-products formed by the reactionof BNBD and Tz). In some examples, as illustrated in FIG. 12A, thereleased moiety Z can be liberated into solution. In other examples, asillustrated in FIG. 12B, Z can remain attached to the carrier moleculeand side product I can be released into solution.

In some examples, bioorthogonal molecules can function as a cleavablelinker between two molecules of interest attached via Z and T. In someexamples, the molecules of interest attached via Z and T can becombinations of biomacromolecules (e.g. oligonucleotide, polypeptide).In some examples, one or more of the modifications (Z or T) can belinked to a surface. In some examples, one or more of the modifications(Z or T) can be linked to a solid support (e.g. nanoparticle, colloid,polymeric support). In some examples, various materials andmacromolecules (e.g. polymers, hydrogels, dendrimers) can includeconnective bioorthogonal molecule elements.

In some examples, compositions can include releasing molecules andbioorthogonal molecules linking two molecules of interest, where contactof the releasing molecule and the bioorthogonal molecule causes thedissociation of the two molecules of interest. One examples of this isillustrated in FIG. 13A. Specifically, a first molecule 162 and a secondmolecule can be bound together via a bioorthogonal molecule. Reaction ofthe bioorthogonal molecule with a releasing group can allow dissociationof the first molecule 162 and the second molecule 164.

In some examples, the two molecules of interest can be polypeptides oroligonucleotides. In the case of oligonucleotides, cleavage may causedissociation of a duplex, as illustrated in FIG. 13B.

In some examples, compositions can include releasing molecules andbioorthogonal molecules where two molecules of interest are attached toa surface or solid support, in which contact of the releasing moleculeand the bioorthogonal molecule causes the cleavage of a molecules ofinterest from a surface or a solid support.

In some examples, compositions can include releasing molecules andmaterials and macromolecules, which include connective bioorthogonalmolecule elements and in which contact of the releasing molecules andthe bioorthogonal molecules with the material/macromolecule leads topartial or complete degradation of the latter, as illustrated in FIG.13C.

In some examples, the bioorthogonal compounds can be used in methods forcontrolling the affinity of a molecule of interest connected by a linkercontaining a bioorthogonal molecule to a target molecule, in whichcleavage of the linker induced by a releasing molecule reduces theaffinity to the target. For example, as illustrated in FIG. 13D, a firstmolecule 162 and a second molecule 164 can be connected by a linkerincluding a bioorthogonal molecule. This can increase the affinity ofthe first molecule 162 and second molecule 164 for the target molecule166. Reaction with a releasing group can release the bioorthogonalmolecule from the linker group to allow dissociation of the firstmolecule 162 and the second molecule 164, which can also decrease theaffinity toward target molecule 166. Thus, the strong binding betweenthe target molecule 166 and the first and second molecules 162, 164 canbe reduced to weak binding therebetween as a result of the release ofthe bioorthogonal molecule from the linker group.

In some examples, the bioorthogonal molecule can be used in methods forcontrolling the activity of an enzyme modified with an inhibitor via alinker containing a bioorthogonal molecule by cleavage of the linker bya releasing molecule, which reduces the inhibition of the enzyme. Forexample, as illustrated in FIG. 13E, an inactive enzyme 167 can bemodified with an inhibitor 169. The inhibitor 169 and the inactiveenzyme 167 can be linked together via a linker including a bioorthogonalmolecule. Reaction with a releasing group can cause the release of thebioorthogonal molecule and the inhibitor 169 to produce an active enzyme168. In some examples, activation of the enzyme can be coupled to areporter event (e.g. bioluminescence, activation of a fluorophore) ortherapeutic event (e.g. prodrug activation).

In some examples, the bioorthogonal molecule can be used in methods forcontrolling the affinity of a molecule including a molecule of interestand an affinity-control molecule, which reduces the affinity to thetarget, connected by a linker including a bioorthogonal molecule to atarget molecule, in which cleavage of the linker by a releasing moleculerestores the affinity of the molecule of interest to a target molecule.For example, as illustrated in FIG. 13F, a target molecule 172 and anaffinity-control molecule 174 can be linked together via a linkerincluding a bioorthogonal molecule. This can decrease the affinity ofthe target molecule 172 to a molecule of interest 176. Reaction of areleasing group with the bioorthogonal molecule can release thebioorthogonal molecule from the linker, which can also release theaffinity-control molecule 174 from the target molecule. This canincrease the affinity of the target molecule 172 for the molecule ofinterest 176.

In some examples, the bioorthogonal molecules can be used in methods forcontrolling the proximity of a molecule of interest and a targetmolecule. The molecule of interest can be linked to a localizationmolecule via a linker containing a bioorthogonal molecule and localizedto a specific site (e.g. anatomical location, intracellular location,surface) on a second molecule. Cleaving the linker with a releasingmolecule can dissociate the molecule of interest and the targetmolecule. For example, as illustrated in FIG. 13G, a target molecule 172can be linked to a localization molecule 174 via a linker including abioorthogonal molecule. The localization molecule 174 can facilitateinteraction between the target molecule 172 and the molecule interest176. Reaction of the bioorthogonal molecule with a releasing group canrelease the bioorthogonal molecule and the localization molecule 174,which can decrease the affinity of the target molecule 172 and themolecule of interest 176 to prevent or minimize further interactiontherebetween. In some examples, the target molecule may modify themolecule of interest.

In some examples, the bioorthogonal molecules can be used in methods forcontrolling the half-life of a molecule of interest. The molecule ofinterest can be linked to a removal molecule, via a linker containing abioorthogonal molecule, in a system (e.g. cell, organism, patient).Cleaving the linker with a releasing molecule can increase the half-lifeof the molecule of interest. For example, as illustrated in FIG. 13H, atarget molecule 182 can be linked to a removal molecule 184 via a linkerincluding a bioorthogonal molecule. Reaction of the bioorthogonalmolecule with a releasing group can remove the bioorthogonal moleculeand the removal molecule 184 to increase the half-life of the targetmolecule 182. In some examples, the removal molecule can target themolecule of interest to the proteasome, or lysosome. In yet additionalexamples, the removal molecule can interact with a molecule that tags itfor removal (e.g. E3 ubiquitin ligase). In still other examples, theremoval molecules can localize to excretory organs.

In some other examples, the molecule of interest can be linked to aretention molecule, via a linker containing a bioorthogonal molecule, ina system (e.g. cell, organism, patient). Cleaving the linker with areleasing molecule can decrease the half-life of the molecule ofinterest and lead to removal of the molecule of interest from thesystem. This can also be illustrated using FIG. 13H, where the molecule184 represents a retention molecule instead of a removal molecule.Reaction of the bioorthogonal molecule with a releasing group can removethe bioorthogonal molecule and the retention molecule 184 to decreasethe half-life of the target molecule 182. In some examples, theretention molecule can be a molecule that enhances the circulatoryhalf-life of a molecule such as serum albumin or a molecule that bindsserum albumin.

In some examples, the bioorthogonal molecule can be used in methods forcontrolling the localization of a molecule of interest linked to alocalization molecule via a linker containing a bioorthogonal moleculeto a specific site (e.g. anatomical location, intracellular location,surface). Cleaving the linker with a releasing molecule can causedissociation of the molecule of interest from the specific site.

In some examples, the bioorthogonal molecule can be used in methods ofimaging one or a series of objects with molecules including a bindingmoiety (e.g. small-molecule, polypeptide, oligonucleotide) and areporter molecule (e.g. fluorophore) connected via a linker containing abioorthogonal molecule. In this example, a first set of molecules canbind to the target molecules, the reporter signal can be measured (e.g.fluorescence microscopy), the reporter molecule can released by cleavageof the linker by contact with a releasing molecule, and delivery of asecond set of molecules for reporting. The cycle can be repeated asdesired.

In some examples, the bioorthogonal molecule can be used in methods forthe spatio-temporal release of molecules, in which the molecules ofinterest are embedded in a material or macromolecule includingconnective bioorthogonal molecule elements and are released upon partialor complete degradation of the material/macromolecule upon contact witha releasing molecule. For example, as illustrated in FIG. 13I, amaterial 184 and associated macromolecules 194 can be bound together viaconnective bioorthogonal molecule elements. Reaction of thebioorthogonal molecule elements with a releasing group can partially orcompletely degrade the material 184 to release individual materialsegments 184 and individual macromolecules 194.

In some examples, bioorthogonal molecules can be used in methods for thespatio-temporal release of molecules, in which the molecules of interestare bound to a molecule consisting of two affinity binders connected bya linker containing a bioorthogonal molecules, which upon contact with areleasing molecule releases the molecule of interest.

In some examples, a bioorthogonal molecule having a structure accordingto Formula V can be used in methods of spatio-temporal controlledrelease of a molecule of interest Z (e.g. therapeutic agent, reportermolecule) by spontaneous decomposition of the bioorthogonal molecule. Insome additional examples, Z can be a therapeutic agent adapted for anydesired means of administration (e.g. orally, intravenously,intravesically, topically, intramuscularly). In some examples, R⁹ canenhance the circulatory half-life of the molecule of interest. In otherexamples, R⁹ can causes the enhanced accumulation of molecules ofinterest at sites of interest (e.g. tissue, tumor, organ).

Various aspects of the bioorthogonal molecules and associatedcompositions, systems, and methods can be illustrated via a number ofnon-limiting examples, as follows:

In some examples, a bioorthogonal molecule, can include a moleculehaving a structure according to:

wherein R¹—R⁸ are independently selected from H, a substituted orunsubstituted C₁-C₄ alkyl or alkylene group, COOH, COOR⁹, COR⁹,CONR⁹R¹⁰, CN, CF₃, and SO₂R⁹, where R⁹ and R¹⁰ are independentlyselected from H and a substituted or unsubstituted C₁-C₄ alkyl oralkylene group, with the proviso that one of R³—R⁸ comprises a leavinggroup, and wherein X is O, S, N, SO, SO₂, SR⁺, Se, PO₂ ⁻, or NRR′⁺, andwhere R and R′ are independently selected from H or a substituted orunsubstituted C₁-C₄ alkyl or alkylene group.

In some examples of a bioorthogonal molecule, X is O or N.

In some examples of a bioorthogonal molecule, R⁵ or R⁸ includes theleaving group.

In some examples of a bioorthogonal molecule, the leaving group iscoupled to R⁵ or R⁸ via a linker group.

In some examples of a bioorthogonal molecule, the linker group is asubstituted or unsubstituted C₁-C₃ alkyl or alkylene group.

In some examples of a bioorthogonal molecule, R⁸ includes the leavinggroup.

In some examples of a bioorthogonal molecule, the leaving group includesa payload selected from the group consisting of a therapeutic agent, aprodrug, a vitamin, a cytotoxic agent, a protein, a nucleic acid, alipid, a polymer, and combinations thereof.

In some examples of a bioorthogonal molecule, the leaving group includesCOOR¹¹, O-Aryl-R¹¹, POR¹¹R¹²R¹³⁺, ONHOR¹¹, or NR¹¹R¹²R¹³⁺, wherein R¹¹,R¹², and R¹³ are independently selected from a payload, H, and asubstituted or unsubstituted C₁-C₄ alkyl or alkylene group.

In some examples of a bioorthogonal molecule, the leaving groupcomprises COOR¹¹ or POR¹¹R¹²R¹³⁺, wherein R¹¹, R¹², and R¹³ areindependently selected from a payload, H, and a substituted orunsubstituted C₁-C₄ alkyl or alkylene group.

In some examples of a bioorthogonal molecule, the molecule has astructure according to:

where Z is the leaving group.

In some examples of a bioorthogonal molecule, R¹, R², or both compriseand electron withdrawing group.

In some examples of a bioorthogonal molecule, the electron withdrawinggroup is a member of the group consisting of COOH, COOR⁹, COR⁹,CONR⁹R¹⁰, CN, CF₃, SO₂R⁹, and NO₂, where R⁹ and R¹⁰ are independentlyselected from H and a substituted or unsubstituted C₁-C₄ alkyl oralkylene group.

In some examples of a bioorthogonal molecule, the bioorthogonal moleculefurther includes a tether group configured to tether the molecule to asubstrate.

In some examples of a bioorthogonal molecule, the tether group isattached to the molecule at one of R³—R⁸ or at X.

In some examples of a bioorthogonal molecule, the tether group isattached to the molecule at X.

In some examples of a bioorthogonal molecule, the substrate is aprotein, a nucleic acid, a lipid, or a polymer.

In some examples of a bioorthogonal molecule, the bioorthogonal moleculefurther includes an SR¹⁴ group coupled to the bioorthogonal molecule atR², wherein R¹⁴ is selected from H or a substituted or unsubstitutedC₁-C₄ alkyl or alkylene group.

In some examples of a bioorthogonal molecule, the biorthogonal moleculeincluding the SR¹⁴ group further includes an electron withdrawing groupcoupled to the molecule at R¹, R², or both.

In some examples of a bioorthogonal molecule, the electron withdrawinggroup is selected from the group consisting of COOH, COOR⁹, COR⁹,CONR⁹R¹⁰, CN, CF₃, and SO₂R⁹, where R⁹ and R¹⁰ are independentlyselected from H and a substituted or unsubstituted C₁-C₄ alkyl oralkylene group.

In some examples of a bioorthogonal molecule, both R¹ and R² includeindividual electron withdrawing groups.

In some examples, a therapeutic composition includes a bioorthogonalmolecule as described herein and a pharmaceutically acceptable carrier.

In some examples of a therapeutic composition, the bioorthogonalmolecule is attached to a carrier molecule.

In some examples of a therapeutic composition, the carrier molecule is amember selected from the group consisting of a protein, aoligonucleotide, a colloid, a nanoparticle, a liposome, a micelle, adendrimer, a surface, a polymer, a viral particle, a cell surface, ahydrogel, a small molecule, and combinations thereof.

In some examples of a therapeutic composition, the pharmaceuticallyacceptable carrier is formulated for administration via injection or isan injectable dosage form.

In some examples of a therapeutic composition, the pharmaceuticallyacceptable carrier is formulated for enteral administration or is anenteral dosage form.

In some examples of a therapeutic composition, the pharmaceuticallyacceptable carrier is formulated as a capsule or tablet.

In some examples of a therapeutic composition, the pharmaceuticallyacceptable carrier is formulated for topical or transdermaladministration or is a topical dosage form or transdermal dosage form.

In some examples of a therapeutic composition, the pharmaceuticallyacceptable carrier is formulated as a gel formulation.

In some examples of a therapeutic composition, the pharmaceuticallyacceptable carrier is formulated as an adhesive patch.

In some examples of a therapeutic composition, the pharmaceuticallyacceptable carrier is formulated for transmucosal administration or is atransmucosal dosage form.

In some examples of a therapeutic composition, the pharmaceuticallyacceptable carrier is formulated as a dissolvable buccal film or tablet.

In some examples of a therapeutic composition, the pharmaceuticallyacceptable carrier is formulated as an eye drop.

In some examples of a therapeutic composition, the therapeuticcomposition further comprises a releasing molecule.

In some examples, a therapeutic system can include a therapeuticcomposition as described herein including a bioorthogonal molecule asdescribed herein and a pharmaceutically acceptable carrier. Thetherapeutic system can also include a releasing composition including areleasing molecule and a second pharmaceutically acceptable carrier.

In some examples of a therapeutic system, the therapeutic composition isdisposed in a first container and the releasing composition is disposedin a second container.

In some examples of a therapeutic system, the bioorthogonal molecule hasa structure according to:

where Z is the leaving group.

In some examples of a therapeutic system, the releasing molecule has astructure according to Formula (III):

where R¹⁵ and R¹⁶ are independently selected from H, 2-pyridine, andPh-CONH((CH₂)₂O)₃Me.

In some examples of a therapeutic system, the releasing molecule has astructure according to Formula (IV):

where R¹⁵ and R¹⁶ are independently selected from H, 2-pyridine, andPh-CONH((CH₂)₂O)₃Me.

In some examples, a method of reversibly modifying a target molecule caninclude removably coupling a bioorthogonal molecule as described hereinto the target molecule and reacting the biorthogonal molecule with areleasing molecule to remove the bioorthogonal molecule from the targetmolecule.

In some examples of a method of reversibly modifying a target molecule,the bioorthogonal molecule is coupled to the target molecule viareaction of the target molecule with a reactive precursor of thebioorthogonal molecule.

In some examples of a method of reversibly modifying a target molecule,the bioorthogonal molecule is incorporated onto the target moleculeduring synthesis of the target molecule.

In some examples of a method of reversibly modifying a target molecule,coupling the biorthogonal molecule to the target molecule inactivatesthe target molecule.

In some examples of a method of reversibly modifying a target molecule,the biorthogonal molecule acts as a protecting group.

In some examples of a method of reversibly modifying a target molecule,the target molecule is a member of the group consisting of apolypeptide, a carbohydrate, a nucleic acid, a lipid, and combinationsthereof.

In some examples, a method of administering a therapeutic agent to asubject can include administering a bioorthogonal molecule as describedherein to the subject, the bioorthogonal molecule having the therapeuticagent releasably coupled thereto. The method also includes reacting thebioorthogonal molecule with a releasing molecule to separate thebioorthogonal molecule from the therapeutic agent.

In some examples of a method of administering a therapeutic agent to asubject, the bioorthogonal molecule is coupled to a carrier molecule.

In some examples of a method of administering a therapeutic agent to asubject, the therapeutic agent is released from the carrier moleculeafter reaction of the bioorthogonal molecule with the releasingmolecule.

In some examples of a method of administering a therapeutic agent to asubject, the therapeutic agent is retained on the carrier molecule afterreaction of the bioorthogonal molecule with the releasing molecule.

In some examples of a method of administering a therapeutic agent to asubject, the releasing molecule is coupled to a carrier molecule.

In some examples of a method of administering a therapeutic agent to asubject, the therapeutic agent is released from the bioorthogonalmolecule after reaction of the bioorthogonal molecule with the releasingmolecule coupled to the carrier molecule.

In some examples of a method of administering a therapeutic agent to asubject, the therapeutic agent is retained on the carrier molecule afterreaction of the bioorthogonal molecule with the releasing moleculecoupled to the carrier molecule.

EXAMPLES Reagents and Methods

As a general overview, all chemical reagents and solvents were obtainedfrom commercial sources (Sigma-Aldrich, Alfa-Aesar, Combi-Blocks,Acros-Organic, TCI) and used without further purification. Mass spectrawere measured by the University of Utah Chemistry Mass SpectrometryFacility. Thin-layer chromatography (TLC) analysis was carried out tomonitor the process of reactions. Purification of compounds was achievedby column chromatography with silica gel 300-400 mesh. ¹H NMR and ¹³CNMR spectra were recorded on a Varian Mercury-400 spectrometer withchemical shifts expressed as ppm (in CDCl₃, MeOD-d₄ or DMSO-d₆) usingMe₄Si (TMS) as internal standard.

Mechanistic studies in DMSO-d₆ were performed by preparing stocksolutions of benzonorbornadiene derivatives 1-3 (24 mM) (See FIG. 15 )and DPTz (24 mM) in DMSO-d₆. Aliquots of the benzonorbornadienederivatives 1-3 stock solution (125 µL) and DPTz stock solution (375 µL)were combined to give final concentrations of 6 mM for the 1-3 and 18 mMfor DPTz. 18-crown-6-ether was added as internal standard for peakintegration. The samples were incubated at 37° C. and ¹H NMR spectrarecorded at the indicated time points (5 min, 30 min, 2 h, 6 h and 24 h)at 25° C.

Mechanistic studies in DMSO-d₆/D₂O (9:1, v/v) were performed bypreparing stock solutions of benzonorbornadiene derivatives 2 (40 mM)and DPTz (24 mM) in DMSO-d₆. Aliquots of 2 stock solution (75 µL), DPTzsolution (375 µL) and D₂O (50 µL) were combined to give finalconcentrations of 6 mM for the 2 and 18 mM for DPTz. 18-crown-6-etherwas added as internal standard for peak integration. The samples wereincubated at 37° C. and ¹H NMR spectra recorded at the indicated timepoints (5 min, 30 min, 2 h, 6 h and 24 h) at 25° C.

Characteristic peaks in ¹H NMR of reaction mixtures were integrated inexperiment performed as described for the ¹H NMR mechanistic studies inDMSO-d₆/D₂O (9:1, v/v). Data was acquired by ¹H NMR monitoring atdifferent time points (5 min, 30 min, 2 h, 6 h and 24 h) in triplicates.

pNA release was quantified by integration of characteristic ¹H NMR peaksin experiments similar to the ¹H NMR mechanistic studies. Aliquots of1-3 stock solution and DPTz solution gave final concentrations of 4.5 mMfor the 1-3 and 18 mM for DPTz (4 eq). 18-crown-6-ether was added asinternal standard for peak integration. The samples were incubated at37° C. and ¹H NMR spectra recorded at the indicated time points (6 h and24 h) at 25° C. The release studies were conducted in triplicate. Theresults are expressed as the mean ± standard deviation (n = 3).

All payload release and stability tests were performed by analyticalreverse-phase HPLC (Thermo Scientific, USA) by using a LUNA C18 column(5 µM, 250×10 mm, Phenomenex, USA). Specifically, stock solutions ofprobes 1-3 (24 mM) and tetrazine (24 mM) in DMSO were prepared. Aliquotsof the benzonorbornadiene derivatives 1-3 stock solution (125 µL) andtetrazine stock solution (375 µL) were combined to give finalconcentrations of 6 mM for the 1-3 and 18 mM for the DPTz. Samples wereincubated at 37° C. and aliquotes were taken at five time points (5 min,30 min, 2 h, 6 h and 24 h) and diluted by 25-fold with MeCN to quenchthe reaction and analyzed by HPLC monitoring. (Blue line: 317 nmchannel; Red line: 378 nm channel)

Release studies of prodrug 5 (See FIG. 15 ) were performed as follows.Stock solutions of prodrug 5 (2 mM) and PEG-Tz (16 mM) in DMSO wereprepared. Aliquots of 5 stock solution (100 µL), Tz stock solution (100µL), DMSO (300 µL) and 0.01 M PBS (500 µL) were combined to give finalconcentrations of 200 µM for the 5 and 1.6 mM for PEG-Tz. The sample wasincubated at 37° C. in the dark and HPLC spectra were recorded at theindicated time points (5 min, 30 min, 2 h and 6 h) at 480 nm.

Stability studies of prodrug 5 in DMSO-PBS (1:1, v/v) were performed asfollows. Stock solutions of prodrug 5 (2 mM) in DMSO were prepared.Aliquots of 5 stock solution (100 µL), DMSO (400 µL) and 0.01 M PBS (500µL) were combined to give final concentrations of 200 µM for the 5 inDMSO-PBS (1:1, v/v). The sample was incubated in the dark at 37° C. andanalyzed by HPLC at the indicated time points (5 min, 6 h and 24 h) at480 nm. No free doxorubicin or doxorubicin-containing side products wereobserved.

Stability studies of prodrug 5 in PBS: Serum were performed as follows.Stock solutions of prodrug 5 (2.5 mM) in DMSO were prepared. Aliquots of5 stock solution (20 µL), 0.01 M PBS (480 µL) and human serum (500 µL)(Sigma-Aldrich, USA) were combined to give final concentrations of 50 µMfor the 5 in PBS: Serum (1: 1, v/v). The sample was thoroughly mixed andincubated at 37° C. in the dark and subsequently a 50 µL aliquot of thesample was taken at indicated time points (5 min, 6 h, 24 h and 48 h)and quenched by 200 µL ice cold acetonitrile, followed by centrifugationat 13300 rpm for 5 min. The supernatant was injected and analyzed byHPLC at 480 nm. No free doxorubicin or doxorubicin-containing sideproducts were observed in the stability test of 5.

Stability studies of compound 2 were performed as follows. Stocksolutions of 2 (2.5 mM) in DMSO were prepared. Aliquots of 2 stocksolution (20 µL), 0.01 M PBS (480 µL) and human serum (500 µL)(Sigma-Aldrich, USA) were combined to give final concentrations of 50 µMfor the 2 in PBS: Serum (1:1, v/v). The sample was thoroughly mixed andincubated at 37° C. in the dark and subsequently a 50 µL aliquot of thesample was taken at indicated time points (5 min, 6 h, 24 h, 48 h, 72 hand 7 days) and quenched by 200 µL ice cold acetonitrile, followed bycentrifugation at 13300 rpm for 5 min. The supernatant was injected andanalyzed by HPLC at 317 nm. No free p-Nitroaniline orp-Nitroaniline-containing side products were observed in the stabilitytest of 2.

UV-VIS photospectrometic kinetic measurements were performed on a BioTekSynergy HT Microplate Reader (BioTek, USA) in a 96-well plate formate.In further detail, for analysis of reaction kinetics in DMSO andDMSO/H₂O (9:1, v/v), stock solutions of benzonorbornadiene derivatives1-3 (60 mM or 12.5 mM) and tetrazine (15 mM or 2.5 mM) in DMSO wereprepared. Final solutions containing tetrazine (2 mM or 0.25 mM) and 1-3(20 mM, 30 mM, 40 mM and 50 mM or 2.5 mM, 3.75 mM, 5 mM, 6.25 mM) wereprepared in 96-well plates and thoroughly mixed at 37° C. for UV-Vismeasurements. For analysis of reaction kinetics in DMSO/PBS (3:2, v/v),final solutions containing tetrazine (0.05 mM) and 1-2 (0.5 mM),tetrazine alone (0.05 mM) were prepared in 96-well plates and thoroughlymixed at 37° C. for UV-Vis measurements. The reactions were monitored at525 nm, which is a local absorbance maximum of tetrazine. All kineticexperiments were run in triplicates. Pseudo-first order curve fittingwas performed with Origin 8.0 software using the exponential formula: y= A₁ × e^(kx) + y₀.

Cell proliferation assays were performed on an Envision 2104 MultilabelReader (PerkinElmer, USA). Specifically, A549 lung cancer cells (ATCC,USA) were maintained in a humidified CO₂ (5%) incubator at 37° C. inDMEM (Thermo Fisher, USA) supplemented with 10% fetal bovine serum inthe presence of 1% Penicillin-Streptomycin-Glutamine (Thermo Fisher,USA) and 0.2% Normocin (InvivoGen, USA). The cells were plated in96-well TC treated plates (PerkinElmer, USA) at a 5000 cells/welldensity 24 h prior to the experiment. Prodrug 5, compound 2 (2 mM inDMSO) and PEG-Tz (40 mM in DMSO) were serially diluted in pre-warmedculture medium. Prodrug 5 and compound 2 are in a series of finalconcentrations ranging from 0.001 to 10 µM with 200 µM, 100 µM, 50 µM,25 µM PEG-Tz before the experiment and added to the wells (100 µL finalvolume per well). Doxorubicin was used as the positive control with sameseries of concentrations ranging from 0.001 to 10 µM. PEG-Tz was alsotested with same series of concentrations ranging from 0.02 to 200 µM,no obvious toxicity was observed. After 72 h incubation at 37° C., cellproliferation was assessed by a CellTiter-Glo® viability assay.Lyophilized CellTiter Glo Substrate (Promega, USA) was dissolved in theCellTiter Glo Buffer to get CellTiter-Glo® Reagent, and from which 100µL was added to each well. After 15 min incubation at 25° C., the mediumwas gently measured with Envision 2104 Multilabel Reader (PerkinElmer,USA) to get the luminescent based on quantitation of the ATP present, anindicator of metabolically active cells. The proliferation assay wasperformed in triplicate (n = 3). EC₅₀ values were derived from thenormalized cell growth and corresponding sigmoidal curves were fittedand generated with Origin 8.0.

Synthetic Reactions

Compounds used in the present example were synthesized as follows:

2-(Trimethylsilyl)phenyl Imidazolsulfonate

2-(Trimethylsilyl)phenyl imidazolsulfonate was prepared as illustratedin FIG. 14A. ¹H NMR (400 MHz, CDCl₃) δ 7.92 (s, 1H), 7.54-7.53 (m, 1H),7.39 (s, 1H), 7.32-7.22 (m, 3H), 6.50 (t, J = 4.4 Hz, 1H), 0.35 (s, 9H).The ¹H NMR data agreed with reported spectra of this compound.

1,4-dihydro-1,4-epoxynaphthalen-1-yl)methanol (1d)

1,4-dihydro-1,4-epoxynaphthalen-1-yl)methanol was prepared according tothe reaction scheme presented in FIG. 14B. In further detail, to asolution of 2-(trimethylsilyl)phenyl imidazolsulfonate (2.37 g, 8.0mmol) and furan-2-ylmethanol (1a; 1.26 g, 12.8 mmol) in anhydrous MeCN(30 mL) was added CsF (2.43 g, 16 mmol). The reaction mixture was heatedand maintained at 50° C. for 8 h. The mixture was extracted with EtOAc(250 mL) and washed with water (2×150 mL). The separated organic layerwas again washed with brine (3×150 mL) and concentrated under reducedpressure. The crude was purified by column chromatography (hexane :EtOAc = 3:1, v/v) to give the desired product as a yellow solid in ayield of 510 mg (36%).

¹H NMR (400 MHz, CDCl₃) δ 7.24-7.22 (m, 1H), 7.18-7.16 (m, 1H), 7.05(dd, J₁ = 1.6 Hz, J₂ = 5.6 Hz, 1H), 6.98-6.96 (m, 2H), 6.88 (d, J = 5.2Hz, 1H), 5.70 (d, J = 1.6 Hz, 1H), 4.48-4.37 (m, 2H), 2.58 (t, d, J =6.0 Hz, 1H). ¹³C NMR (100 MHz, CDCl₃) δ 150.5, 147.8, 144.9, 142.4,125.2, 125.1, 120.2, 119.6, 93.7, 82.2, 60.3. HRMS (ESI): calcd. forC₁₁H₁₀O₂ [M+Na]⁺ 197.0578, found 197.0583.

1,4-dihydro-1,4-epoxynaphthalen-1-yl)methyl (4-nitrophenyl)carbamate (1)

1,4-dihydro-1,4-epoxynaphthalen-1-yl)methyl (4-nitrophenyl)carbamate wasprepared according to the reaction scheme presented in FIG. 14C. Infurther detail, to a solution of 1d (556 mg, 3.2 mmol) and DMAP (507 mg,4.16 mmol) in anhydrous THF (40 mL) was added 4-nitrophenyl isocyanate(1.3 g, 8.0 mmol). The reaction mixture was stirred at 50° C. for 12 h.The mixture was diluted with EtOAc (150 mL) and washed with water (2×100mL) and brine (3×150 mL). The separated organic layer was dried withNa₂SO₄ and concentrated under reduced pressure. The crude compound waspurified by column chromatography (hexane : EtOAc = 3:1, v/v) to affordthe desired compound as a yellow solid in a yield of 180 mg (29%).

¹H NMR (400 MHz, CDCl₃) δ 8.21 (d, J = 9.2 Hz, 2H), 7.55 (d, J = 8.8 Hz,2H), 7.31-7.28 (m, 1H), 7.22-7.20 (m, 1H), 7.16-7.13 (m, 2H), 7.03-7.01(m, 2H), 6.89 (d, J= 5.6 Hz, 1H), 5.77 (d, J = 1.6 Hz, 1H), 5.16 (d, J =12.8 Hz, 1H), 5.02 (d, J = 13.2 Hz, 1H). ¹³C NMR (100 MHz, CDCl₃) δ152.4, 149.8, 147.1, 145.3, 143.5, 141.6, 125.5, 125.3, 125.2, 120.5,119.4, 117.8, 91.1, 82.4, 62.4. HRMS (ESI): calcd. for C₁₈H₁₄N₂O₅[M+Na]⁺ 361.0800, found 361.0804.

1-acetyl-1H-pyrrole-2-carbaldehyde

1-acetyl-1H-pyrrole-2-carbaldehyde was prepared as illustrated in thescheme represented in FIG. 14D.

¹H NMR (400 MHz, CDCl₃) δ 10.29 (s, 1H), 7.33 (dd, J₁ = 1.6 Hz, J₂ = 2.8Hz, 1H), 7.21 (dd, J₁ = 1.6 Hz, J₂ = 4.0 Hz, 1H), 6.35 (t, J = 3.2 Hz,1H), 2.66 (s, 3H). The ¹H NMR data agreed with reported spectra of thiscompound.

1-(hydroxymethyl)-1H-pyrrol-1-yl)ethanone (2a)

1-(hydroxymethyl)-1H-pyrrol-1-yl)ethanone (2a) was prepared asillustrated in the scheme represented in FIG. 14E.

¹H NMR (400 MHz, CDCl₃) δ 7.06 (d, J₁ = 1.6 Hz, J₂ = 3.2 Hz, 1H),6.21-6.19 (m, 2H), 4.61 (s, 2H), 2.57 (s, 3H). ¹³C NMR (100 MHz, CDCl₃)δ 171.5, 135.4, 121.7, 114.7, 112.2, 57.8, 23.7. The ¹H NMR data agreedwith reported spectra of this compound.

1-(((tert-butyldimethylsilyl)oxy)methyl)-1H-pyrrol-1-yl)ethanone (2b)

1-(((tert-butyldimethylsilyl)oxy)methyl)-1H-pyrrol-1-yl)ethenone wasprepared according to the reaction scheme illustrated in FIG. 14F. Infurther detail, to a solution of 2a (1.2 g, 8.6 mmol) and imidazole (877mg, 12.9 mmol) in anhydrous DMF (10 mL) was addedtert-butylchlorodimethylsilane (1.52 g, 10.3 mmol) at 0° C. The reactionmixture was warmed to room temperature and kept at this temperature for3 h. The reaction was quenched with sat. aq. NaHCO₃ solution (200 mL),diluted with water (150 mL) and extracted with EtOAc (2×150 mL). Thecombined organic layers were washed with brine (150 mL), dried overNa₂SO₄ and concentrated under reduced pressure. The crude product waspurified by column chromatography (hexane : EtOAc = 20:1, v/v) to givethe product as a brown oil in a yield of 1.25 g (58%).

¹H NMR (400 MHz, CDCl₃) δ 7.06-7.04 (m, 1H), 6.31-6.30 (m, 1H), 6.22 (t,J= 3.2 Hz, 1H), 4.94 (brs, 2H), 2.53 (s, 3H), 0.93 (s, 9H), 0.09 (s,6H). ¹³C NMR (100 MHz, CDCl₃) δ 169.0, 136.9, 120.3, 112.0, 111.5, 60.7,25.9, 23.5, 18.4, -5.4. HRMS (ESI): calcd. for C₁₃H₂₃NO₂Si [M+Na]⁺276.1396, found 276.1398.

1-(((Tert-butyldimethylsilyl)oxy)methyl)-1,4-dihydro-1,4-epiminonaphthalen-9-yl)ethanone(2c)

1-(((tert-butyldimethylsilyl)oxy)methyl)-1,4-dihydro-1,4-epiminonaphthalen-9-yl)ethenonewas prepared according to the reaction scheme illustrated in FIG. 14G.In further detail, to a solution of 2-(trimethylsilyl)phenylimidazolsulfonate (0.98 g, 3.3 mmol) and 2b (1.25 g, 5 mmol) inanhydrous MeCN (20 mL) was added CsF (1 g, 6.6 mmol). The reactionmixture was heated and maintained at 60° C. for 12 h. The mixture wasdiluted with EtOAc (150 mL) and washed with water (2×50 mL). Theseparated organic layer was again washed with brine (2×150 mL) andconcentrated under reduced pressure. The crude was purified by columnchromatography (hexane : EtOAc = 2:1, v/v) to give the product as abrown oil in a yield of 350 mg (32%).

¹H NMR (400 MHz, CDCl₃) δ 7.46 (d, J = 8.0 Hz, 1H), 7.21 (d, J = 8.0 Hz,1H), 7.14 (brs, 1H), 7.00-6.93 (m, 3H), 5.41 (s, 1H), 4.96 (d, J = 8.0Hz, 1H), 4.68 (d, J = 8.0 Hz, 1H), 1.93 (s, 3H), 0.96 (s, 9H), 0.21 (s,3H), 0.19 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 167.5, 151.1, 148.5,145.5, 141.2, 125.2, 124.7, 121.2, 119.7, 77.1, 67.1, 60.9, 25.9, 22.7,18.2, -5.3, -5.4, -5.5. HRMS (ESI): calcd. for C₁₉H₂₇NO₂Si [M+Na]⁺352.1709, found 352.1716.

1-(Hydroxymethyl)-1,4-dihydro-1,4-epiminonaphthalen-9-yl)ethanone (2d)

1-(hydroxymethyl)-1,4-dihydro-1,4-epiminonaphthalen-9-yl)ethenone wasprepared according to the reaction scheme illustrated in FIG. 14H. Infurther detail, to a solution of 2c (350 mg, 1.1 mmol) in THF (4 mL) wasadded 1 M tetra n-butyl ammonium fluoride (2 mL. 2 mmol) at roomtemperature, and the mixture was stirred for 2 h. The mixture wasdiluted with Et₂O/EA mixture (100 mL, 1:1) and washed with water (50 mL)and brine (2×50 mL) and the separated organic layer was dried withNa₂SO₄, and concentrated under reduced pressure. The crude product waspurified by column chromatography (hexane : EtOAc = 10:1, v/v) to affordthe product as a brown oil in a yield of 200 mg (85%).

¹H NMR (400 MHz, CDCl₃) δ 7.29-7.26 (m, 2H), 7.08-7.06 (m, 1H),7.04-7.00 (m, 2H), 6.93 (d, J= 5.6 Hz, 1H), 5.51 (d, J = 2.4 Hz, 1H),5.30-5.26 (m, 1H), 4.59 (dd, J₁ = 3.2 Hz, J₂ = 7.2 Hz, 2H), 2.05 (s,3H). ¹³C NMR (100 MHz, CDCl₃) δ 164.8, 148.2, 147.8, 145.4, 143.3,125.5, 125.3. 120.5, 120.2, 79.0, 66.8, 58.0, 22.3. HRMS (ESI): calcd.for C₁₃H₁₃NO₂ [M+Na]⁺ 238.0844, found 238.0842.

9-Acetyl-1, 4-dihydro-1, 4-epiminonaphthalen-1-yl)methyl(4-nitrophenyl)carbamate (2)

9-acetyl-1,4-dihydro-1,4-epiminonaphthalen-1-yl)methyl(4-nitrophenyl)carbamate was prepared according to the reaction schemeillustrated in FIG. 14I. In further detail, to a solution of 2d (200 mg,0.9 mmol) and DMAP (146 mg, 1.2 mmol) in anhydrous THF (12 mL) was added4-nitrophenyl isocyanate (300 mg, 2 mmol). The reaction mixture wasstirred at 50° C. for 10 h. The mixture was diluted with EtOAc (150 mL)and washed with water (200 mL) and brine (2×150 mL). The separatedorganic layer was dried with Na₂SO₄, and concentrated under reducedpressure, purified by column chromatography (hexane: EtOAc = 5:1, v/v)to afford the desired compound as yellow solid in a yield of 70 mg(22%).

¹H NMR (400 MHz, DMSO-d₆) δ 10.50 (s, 1H), 8.22 (d, J = 9.6 Hz, 2H),7.76 (d, J= 9.6 Hz, 2H), 7.37-7.35 (m, 2H), 7.18-7.16 (m, 1H), 7.06 (d,J = 5.6 Hz, 1H), 7.01-6.99 (m, 2H), 5.84 (d, J = 2.4 Hz, 1H), 5.40-5.30(m, 2H), 1.89 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 169.3, 153.6, 149.7,149.3, 146.1, 144.9, 143.9, 142.2, 125.5, 125.2, 121.0, 120.6, 118.2,75.3, 67.0, 62.2, 23.4. HRMS (ESI): calcd. for C₂₀H₁₇N₃O₅ [M+Na]⁺402.1066, found 402.1075.

Tert-butyl 2-formyl-1H-pyrrole-1-carboxylate

Tert-butyl 2-formyl-1H-pyrrole-1-carboxylate was prepared as illustratedin the scheme depicted in FIG. 14J.

¹H NMR (400 MHz, CDCl₃) δ 10.23 (s, 1H), 7.34 (brs, 1H), 7.06 (brs, 1H),6.18 (t, J = 4.0 Hz, 1H), 1.55 (s, 9H). The ¹H NMR data agreed withreported spectra of this compound.

Tert-butyl 2-(hydroxymethyl)-1H-pyrrole-1-carboxylate (3a)

Tert-butyl 2-(hydroxymethyl)-1H-pyrrole-1-carboxylate (3a) was preparedas illustrated in the scheme depicted in FIG. 14K.

¹H NMR (400 MHz, CDCl₃) δ 7.16 (d, J = 1.2 Hz, 1H), 6.18 (brs, 1H), 6.10(brs, 1H), 4.64 (d, J = 7.6 Hz, 2H), 3.60 (d, J = 7.2 Hz, 1H), 1.61 (s,9H). ¹³C NMR (100 MHz, CDCl₃) δ 150.0, 134.8, 121.9, 113.6, 110.4, 84.5,57.7, 28.0. The ¹H NMR data agreed with reported spectra of thiscompound.

Tert-butyl2-(((tert-butyldimethylsilyl)oxy)methyl)-1H-pyrrole-1-carboxylate (3b)

Tert-butyl2-(((tert-butyldimethylsilyl)oxy)methyl)-1H-pyrrole-1-carboxylate wasprepared according to the reaction scheme illustrated in FIG. 14L. Infurther detail, to a solution of 3a (7.0 g, 35.5 mmol) and imidazole(3.6 g, 53 mmol) in anhydrous DMF (30 mL) was addedtert-butylchlorodimethylsilane (6.5 g, 44 mmol) at 0° C. The reactionmixture was allowed to warm to room temperature and stirred for 12 h.The reaction mixture was extracted with DCM (300 mL), quenched with sat.aq. NaHCO₃ solution (150 mL), and washed with water (2×200 mL). Thecombined organic layers were washed with brine (2×200 mL), dried overNa₂SO₄ and concentrated under reduced pressure. The crude product waspurified by column chromatography (hexane : EtOAc = 20:1, v/v) to givethe product as a brown oil in a yield of 8.8 g (80%).

¹H NMR (400 MHz, CDCl₃) δ 7.19 (t, J = 2.4 Hz, 1H), 6.23 (brs, 1H), 6.13(t, J= 3.2 Hz, 1H), 4.89 (s, 2H), 1.59 (s, 9H), 0.93 (s, 9H), 0.09 (s,6H). ¹³C NMR (100 MHz, CDCl₃) δ 149.2, 135.4, 120.9, 111.0, 110.3, 83.5,60.2, 27.9, 25.9, 18.4, -5.4. HRMS (ESI): calcd. for C₁₆H₂₉NO₃Si [M+Na]⁺334.1814, found 334.1815.

Tert-butyl-1-(((tert-butyldimethylsilyl)oxy)methyl)-1, 4-dihydro-1,4-epiminonaphthalene-9-carboxylate (3c)

tert-butyl-1-(((tert-butyldimethylsilyl)oxy)methyl)-1,4-dihydro-1,4-epiminonaphthalene-9-carboxylatewas prepared according to the reaction scheme illustrated in FIG. 14M.In further detail, to a solution of 2-(trimethylsilyl)phenylimidazolsulfonate (4.5 g, 15 mmol) and 3b, (7.2 g, 22.8 mmol) inanhydrous MeCN (60 mL) was added CsF (4.7 g, 30 mmol). The reaction wasmaintained at 60° C. for 12 h. The mixture was diluted with EtOAc (150mL) and washed with water (100 mL). The separated organic layer waswashed with brine (2×100 mL), dried with Na₂SO₄ and concentrated byreduced pressure. The crude was purified by column chromatography(hexane : EtOAc = 100:1 to 50:1, v/v) to give the product as a brown oilin a yield of 1.2 g (20%).

¹H NMR (400 MHz, CDCl₃) δ 7.42 (d, J = 6.0 Hz, 1H), 7.22 (d, J = 6.0 Hz,1H), 7.06 (d, J = 5.2 Hz, 1H), 6.95 (t, J = 6.0 Hz, 3H), 5.44 (s, 1H),4.78 (brs, 1H), 4.55 (d, J = 9.6 Hz, 1H), 1.32 (s, 9H), 0.96 (s, 9H),0.20 (s, 3H), 0.19 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 154.8, 150.5,149.5, 144.4, 141.6, 141.5, 124.9, 124.6, 120.9, 120.8, 120.5, 80.5,67.4, 61.4, 29.7, 28.1, 25.9, 18.3, -5.3, -5.4. HRMS (ESI): calcd. forC₂₂H₃₃NO₃Si [M+Na]⁺ 410.2127, found 410.2138.

Tert-butyl1-(hydroxymethyl)-1,4-dihydro-1,4-epiminonaphthalene-9-carboxylate (3d)

tert-butyl-1-(hydroxymethyl)-1,4-dihydro-1,4-epiminonaphthalene-9-carboxylatewas prepared according to the reaction scheme illustrated in FIG. 14N.In further detail, to a solution of 3c (1.1 g, 3.0 mmol) in anhydrousTHF (7 mL) was added 1 M tetra n-butyl ammonium fluoride (4.8 mL, 4.8mmol) at room temperature and the mixture was stirred for 8 h. Themixture was extracted with Et₂O/EtOAc mixture (50 + 50 mL) and washedwith water (2×50 mL). The separated organic layer was again washed withbrine (2×50 mL), dried with Na₂SO₄ and concentrated under reducedpressure. The crude compound was purified by column chromatography(hexane : EtOAc = 10:1, v/v) to afford the product as a brown oil in ayield of 0.7 g (85%).

¹H NMR (400 MHz, CDCl₃) δ 7.24 (t, J = 5.2 Hz, 2H), 7.04-6.98 (m, 3H),6.88 (d, J = 5.2 Hz, 1H), 5.51 (brs, 1H), 4.58 (d, J = 6.8 Hz, 2H), 1.42(s, 9H). ¹³C NMR (100 MHz, CDCl₃) δ 148.8, 148.1, 144.4, 144.0, 125.1,125.0, 120.5, 120.1, 81.6, 78.6, 66.9, 58.9, 28.2, 25.6. HRMS (ESI):calcd. for C₁₆H₁₉NO₃ [M+Na]⁺ 296.1263, found 296.1267.

Tert-butyl1-((((4-nitrophenyl)carbamoyl)oxy)methyl)-1,4-dihydro-1,4-epiminonaphthalene-9-carboxylate(3)

tert-butyl-1-((((4-nitrophenyl)carbamoyl)oxy)methyl)-1,4-dihydro-1,4-epiminonaphthalene-9-carboxylatewas prepared according to the reaction scheme illustrated in FIG. 140 .In further detail, to a solution of 3d (408 mg, 1.5 mmol) and DMAP (244mg, 2 mmol) in THF (25 mL) was added 4-nitrophenyl isocyanate (450 mg, 3mmol). The reaction was stirred at 50° C. for 10 h. The mixture wasdiluted with EtOAc (150 mL) and washed with water (200 mL) and brine(2×150 mL). The separated organic layer was dried with Na₂SO₄,concentrated under reduced pressure and purified by columnchromatography (hexane : EtOAc = 5 :1, v/v) to afford the desiredcompound as a yellow solid in a yield of 405 mg (62%).

¹H NMR (400 MHz, CDCl₃) δ 8.21 (d, J = 9.2 Hz, 2H), 7.57 (d, J = 9.2 Hz,2H), 7.31-7.29 (m, 1H), 7.24-7.22 (m, 2H), 7.08-7.06 (m, 1H), 7.03-6.98(m, 2H), 6.84 (d, J= 5.6 Hz, 1H), 5.55 (d, J = 2.0 Hz, 1H), 5.40 (brs,2H), 1.36 (s, 9H). ¹³C NMR (100 MHz, CDCl₃) δ 154.8, 152.6, 148.6,147.9, 143.9, 142.9, 126.3, 125.4, 125.2, 125.1, 121.1, 119.8, 117.7,81.4, 75.6, 67.4, 62.2, 28.1. HRMS (ESI): calcd. for C₂₃H₂₃N₃O₆ [M+Na]⁺460.1485, found 460.1495.

Doxorubicin-Containing Prodrug (5)

The doxorubicin-containing prodrug was prepared according to thereaction scheme illustrated in FIG. 14P. In further detail, to asolution of 2d (53 mg, 0.24 mmol) in dry DCM (6 mL) was added DMAP (0.5mmol, 70 mg) and nitrophenyl chloroformate (71 mg, 0.35 mmol) at 0° C.The reaction was kept in the dark at 25° C. overnight. The reactionmixture was quenched with ice and extracted with DCM (2×20 mL). Thecombined organic layer was washed with water (3×50 mL) and brine (3×50mL) until no more yellow color was observed in the organic phase, driedover Na₂SO₄ and concentrated to afford the carbonate intermediate (4) aslight yellow solid in a yield of 65 mg (64%). 4 decomposed upon storageand was immediately used in the next step.

¹H NMR (400 MHz, CDCl₃) δ 7.29 (dd, J₁ = 2.0 Hz, J₂ = 6.8 Hz, 2H), 7.44(dd, J₁ = 2.0 Hz, J₂ = 6.8 Hz, 2H), 7.31-7.26 (m, 2H), 7.10-7.08 (m,1H), 7.04-6.99 (m, 3H), 5.65-5.62 (m, 1H), 5.67 (s, 1H), 5.53 (d, J =7.6 Hz, 1H), 1.99 (s, 3H).

To a solution of 4 (64 mg, 0.17 mmol) in dry DMF (0.5 mL) was added DIEA(272 mg, 2.1 mmol), after 15 min, doxorubicin hydrochloride (120 mg, 0.2mmol) was added and the reaction mixture was stirred in the dark and at25° C. for 24 h. The mixture was diluted with DCM (100 mL) and washedwith H₂O (50 mL) and brine (2×50 mL). The organic phase was concentratedand purified by preparatory-TLC (DCM : MeOH = 15:1, v/v) to afford thedesired compound as red solid in a yield of 40 mg (30%).

¹H NMR (400 MHz, CDCl₃:MeOD-d₄ = 9:1) δ 7.97 (dd, J₁ = 1.2 Hz, J₂ = 8.0Hz, 1H), 7.73 (t, J = 8.0 Hz, 1H), 7.34 (d, J = 8.4 Hz, 1H), 7.17 (brs,2H), 6.95-6.90 (m, 2H), 6.81 (brs, 1H), 6.74 (brs, 1H), 5.42 (brs, 2H),5.23 (d, J = 15.2 Hz, 2H), 5.11 (d, J = 11.6 Hz, 1H), 4.71 (s, 2H), 4.08(d, J = 6.4 Hz, 1H), 4.01 (s, 3H), 3.79 (d, J = 12.0 Hz, 1H), 3.62 (s,1H), 3.20 (d, J = 18.8 Hz, 1H), 2.98 (d, J = 18.8 Hz, 1H), 2.30 (d, J =15.2 Hz, 1H), 2.09 (d, J = 15.2 Hz, 1H), 1.89 (d, J = 4.0 Hz, 3H),1.80-1.74 (m, 2H), 1.23 (d, J = 6.4 Hz, 3H). ¹³C NMR (126 MHz, DMSO-d₆)δ 214.3, 186.9, 186.8, 169.3, 161.2, 156.6, 155.7, 154.9, 150.3, 144.7,143.5, 136.6, 135.9, 135.1, 134.5, 125.3, 125.1, 120.9, 120.6, 120.4,120.2, 119.4, 111.2, 111.0, 100.8, 75.4, 70.3, 68.4, 67.1, 67.0, 64.2,61.3, 57.0, 55.4, 47.7, 37.0, 32.5, 30.3, 23.2, 17.5. HRMS (ESI): calcd.for C₄₁H₄₀N₂O₁₄ [M+Na]⁺ 807.2377, found 807.2383.

3,6-Di(pyridin-2-yl)pyridazine (DPPz)

DPPz was prepared according to the reaction scheme illustrated in FIG.14Q. In further detail, to a solution of 1,4-epoxynaphthalene (72 mg,0.5 mmol) in MeCN (12 mL) was added a solution of3,6-di(pyridin-2-yl)-1,2,4,5-tetrazine (118 mg, 0.5 mmol) in MeCN (12mL). The reaction was stirred at 40° C. for 4 h. The mixture wasconcentrated and purified by column chromatography (hexane : EtOAc =1:1, v/v) to afford the desired compound as a pale solid in a yield of60 mg (49%).

¹H NMR (400 MHz, DMSO-d₆) δ 8.80 (d, J = 3.6 Hz, 2H), 8.69 (s, 2H), 8.65(d, J = 6.4 Hz, 2H), 8.08 (t, J = 6.4 Hz, 2H), 7.61-7.59 (m, 2H). The ¹HNMR agreed with reported spectra of this compound.

4-(1,2,4,5-Tetrazin-3-yl)benzoic Acid

4-(1,2,4,5-tetrazin-3-yl)benzoic acid was synthesized as illustrated inthe scheme depicted in FIG. 14R.

¹H NMR (400 MHz, DMSO-d₆) δ 10.65 (s, 1H), 8.64-8.57 (m, 2H), 8.24-8.17(m, 2H). The ¹H NMR data agreed with reported spectra of this compound.

N-methoxyethoxy)ethoxy)ethyl)-4-(1,2,4,5-tetrazin-3-yl) benzamide(PEG-Tz)

PEG-Tz was prepared according to the reaction scheme illustrated in FIG.14S. In further detail, to a dry round-bottom flask was added2-(2-(2-methoxyethoxy)ethoxy)ethan-1-amine (190 mg, 1.19 mmol) in THF(0.5 mL), dicyclohexylmethanediimine (230 mg, 1.09 mmol) in THF (1.0mL), and hydroxybenzotriazole (170 mg, 1.09 mmol) in THF (1 mL). Thesolution was cooled to 0° C. in an ice bath and stirred.4-(1,2,4,5-tetrazin-3-yl)benzoic acid (200 mg, 0.99 mmol) in THF (2.5mL) was added to the flask while allowing the reaction mixture to returnto room temperature and the resultant red reaction mixture was stirredfor 20 h. After the allotted time, the reaction was diluted with 10 mLof diethyl ether to precipitate out the dicyclohexylurea byproduct,which was removed by filtration; the solution was subsequently washedwith diethyl ether (2×5 mL), filtered again, and concentrated. Themixture was directly concentrated and purified by column chromatography(DCM : Acetone = 10:1, v/v) to afford the desired compound as a pinksolid in a yield of 80 mg (20%)

¹H NMR (400 MHz, CDCl₃) δ 10.27 (s, 1H), 8.75-8.67 (m, 2H), 8.09-8.01(m, 2H), 7.00 (s, 1H), 3.77-3.62 (m, 9H), 3.58-3.51 (m, 2H), 3.33 (s,3H). ¹³C NMR (100 MHz, CDCl₃) δ 166.4, 165.9, 157.9, 138.7, 134.0,128.4, 128.1, 71.9, 70.6, 70.5, 70.2, 69.7, 39.9. HRMS (ESI): calcd. forC₁₆H₂₁N₅O₄ [M+Na]⁺ 370.1491, found 370.1496.

Results

Benzonorbornadiene (BNBD) derivatives are described in this example asstable carrier molecules that rapidly react with Tz to quantitativelyrelease a cargo molecule (e.g. cytotoxic agent, optical reporter). Thismolecular design is based on the intrinsic lability of isobenzofurans,isoindoles, and the like, which can be harnessed to near-instantaneouslyliberate a cargo molecule. Further, such self-immolative heterocyclesare readily accessible from the reaction of BNBDs and Tz. In particular,the proposed release molecules can include 7-aza/oxa-BNBDs withcarbamate leaving groups, or other suitable leaving group as describedabove, attached via a methylene linker, or other suitable linker asdescribed above, to the bridgehead carbon (See FIG. 15 for Scheme 1).For example, the reaction of 7-aza/oxa-BNBDs with Tz generates anintermediate (I1), which rapidly eliminates N₂, followed by a retroDiels-Alder cycloreversion, generating isoindoles/isobenzofurans (I3).These heterocycles in turn eliminate a carbamate to liberate the freeamine (Scheme 1a). In contrast to the carbamate intermediate I3, theBNBD precursors are expected to be highly stable. Several BNBD-releasemolecules were successfully synthesized and demonstrated to undergo arapid and traceless cargo-release reaction with Tz. Payload liberationwas near-quantitative, and isoindole intermediates decomposed so rapidlythat they were undetectable. Cytotoxicity assays demonstrated theefficient restoration of doxorubicin from a BNBD-prodrug, whereas theprodrug itself showed little toxicity. Importantly, BNBD molecules werehighly stable and no unspecific payload release was observed after aweek of incubation in human serum.

With this overview in mind, benzonorbornadiene (BNBD) derivatives weredesigned to release a drug or reporter molecule upon reaction withtetrazine (Tz), as illustrated in FIG. 15 (Scheme 1).

In further detail, to test the proposed molecular design, three BNBDderivatives were synthesized with oxygen (1), acetamide (2), andBoc-protected nitrogen (3) at position 7 of the BNBD bicycle and a(p-nitrophenylcarbamoyl)methyl substituent at the bridgehead carbon(Scheme 1b). The different substituents in 1-3 were selected to test theinfluence of the heterocycle on reaction rate and cargo release.p-Nitroaniline (pNA) was selected as the reporter for bond-cleavagebecause a bathochromic shift of the absorbance maximum from λ_(max) =317 nm to λ_(max) = 378 nm accompanies conversion from a carbamate to anamine. The synthesis of 1-3 (Scheme 1b) started from furfuryl alcohol(1a) and N-acetyl- or N-Boc-(2-hydroxymethyl)pyrroles (2a,3a). A [4+2]cycloaddition reaction of these heterocylces with benzyne afforded thebicyclic structures 1d-3d. The pyrroles required TBDMS-protection of thehydroxymethyl group (2b,3b) for the benzyne reaction. Reaction ofhydroxymethyl bicycles 1d-3d with (4-nitrophenyl)isocyanate provided thedesired release structures 1-3.

It was first investigated whether the reaction of BNBD-derivatives withTz released pNA (FIGS. 16A-16B). Specifically, stock solutions of 1 (12mM) and DPTz (12 mM) in DMSO were prepared. Aliquots of the DPTz stocksolution (500 µL) was added with DMSO (500 µL); Solution of 1 (500 µL)was added with DPTz stock solution (500 µL); Solution of 1 (500 µL) wasadded with DMSO (500 µL). The samples were incubated at 37° C. and theimage was recorded at the time point of 24 h, shown as FIG. 16A.

Incubation of 1-3 with 3,6-di-(2-pyridyl)-1,2,4,5-tetrazine (DPTz)resulted in a distinct color change from pink to yellow (vials from leftto right in FIG. 16A), which indicated complete DPTz consumption andefficient pNA release. Control samples with 1-3 or DPTz alone exhibitedno color change over >24 h, and ¹H NMR and HPLC analysis confirmed thestability of all individual reagents (data not shown). HPLC monitoringof the reaction (c(1-3) = 6 mM, c(DPTz) = 18 mM, T = 37° C., DMSO/H₂O(9:1)) confirmed complete consumption of 1-3, disappearance of DPTz, andformation of two new elution peaks that were assigned as pNA and3,6-di-(2-pyridyl)-1,2-pyridazine (DPPz) based on retention time,extinction maxima of absorbance spectra, and molecular mass (FIGS.16B-16E). Importantly, the starting BNBD was the only detectable peakwith an absorbance maximum at λ_(max) = 317 nm (FIG. 16B) andintermediates I1-I3 or side-products with trapped pNA were not observed.Products of isoindole/isobenzofuran decomposition were visible in theHPLC traces at later measurement times (FIG. 16B). This outcomedemonstrated that the release of the amine from the isoindoleintermediate I3 is rapid and high-yielding.

Having established the Tz-induced release of pNA, the kinetics of thereaction of 1-3 and Tz were measured (See FIGS. 22A-22K).Photospectrometric analysis of DPTz disappearance (λ_(abs) = 525 nm) inthe presence of excess BNBDs revealed pseudo-first order kinetics, andthe concentration dependence of the rate constants agreed with asecond-order rate law. In DMSO, the second order rate constants (k₂)were 0.015 M⁻¹ s⁻¹ for 1, 0.010 M⁻¹ s⁻¹ for 2, and 0.0044 M⁻¹ s⁻¹ for 3(Table 1). The differences in the rate constants reflect the increase insteric repulsion from 1 to 3 although electronic effects may alsoinfluence the reaction rate. Comparison of the kinetics of the reactionof DPTZ with 1 to that with 7-oxo-BNBD (6, Scheme 1b; k₂ = 0.176 M⁻¹s⁻¹) revealed that the (p-nitrophenylcarbamoyl)methyl substituentdecreased the reaction rate ~12-fold. Presence of 10% water acceleratedthe reaction 1.7 to 1.9-fold (Table 1; 1: k₂ = 0.028 M⁻¹ s⁻¹; 2: k₂ =0.017 M⁻¹ s⁻¹; 3: k₂ = 0.0084 M⁻¹ s⁻¹). This result is in agreement withreported rate-enhancing effects of water on IEDDA reactions. Awater-soluble Tz (PEG-Tz; Scheme 1a) was also prepared and tested toevaluate the solvent effect on the reaction kinetics (Table 1).Increasing the water content further (DMSO/PBS, 3:2) substantiallyaccelerated the reaction rate of 2 (k₂ = 0.135 M⁻¹ s⁻¹) and 1 (k₂ =0.190 M⁻¹ s⁻¹) with PEG-Tz. These reaction rates were comparable tothose of TCO-based release molecules with 4,6-dimethyltetrazine (k₂ =0.54 M⁻¹ s⁻¹). More electron-deficient Tz molecules reacted faster withTCO-prodrugs but at the expense of incomplete drug release. The reactionof 1 and 2 with Tz was significantly faster than the release reaction ofTCO with aromatic azides (k₂ = 0.027 M⁻¹ s⁻¹), Tz-induced uncaging ofvinyl ethers (k₂ = 0.00021 M⁻¹ s⁻¹), and the Staudinger reaction withrelease functionality (k₂ = ~0.001 M⁻¹ s⁻¹). In conclusion, the reactionof BNBD with Tz proceeds at a comparable or higher rate than previousbioorthogonal release reactions.

TABLE 1 - Second-order rate constants (k₂) for reactions of 1-3 andtetrazines (M⁻¹s⁻¹)^(a) Pb Tz DMSO 90%DMSO/H₂O 60%DMSO/PBS 1 DPTz 0.015± 0.008 0.028 ± 0.0003 n/a 2 DPTz 0.010 ± 0.0004 0.017 ± 0.002 n/a 3DPTz 0.0044 ± 0.00009 0.0084 ± 0.0016 n/a 6 DPTz 0.176 ± 0.004 n/a n/a 1PEG-Tz n/a 0.058 ± 0.0003 0.190 ± 0.029 2 PEG-Tz n/a 0.020 ± 0.00070.135 ± 0.010 ^(a) The reactions were monitored by time-dependentabsorbance measurements at λ_(abs) = 525 nm and T = 37° C.

To quantify pNA release and to analyze the mechanism of the reaction of1-3 and DPTz, the transformation was monitored in a series of ¹H NMRexperiments (FIGS. 17A-17G). Solutions of 1-3 and DPTz in DMSO-d₆ orDMSO-d₆/D₂O (9:1) were incubated at 37° C. and ¹H NMR spectra wererecorded periodically over 24 h. At these conditions, DPTznear-completely consumed BNBD derivatives within 2 h in agreement withresults from HPLC analysis (FIG. 16B) and kinetics measurements (Table1). NMR peaks corresponding to pNA emerged concomitant withdisappearance of 1-3, indicating rapid and efficient cargo liberation(FIGS. 17A-17G). The measured pNA release from 1 and 2 in DMSO-d₆/D₂O at6 h and 24 h was in the range of 80-90%. Release of pNA from 3 was lessefficient especially in water-free DMSO-d₆ (FIG. 17B).

Analysis of ¹H NMR integrations revealed that in the case of 1 theformation of pNA was delayed relative to BNBD consumption (FIG. 17D). Asinglet peak at 5.59 ppm was present in early ¹H NMR spectra butdisappeared with longer incubation times. Integration of this peakaccounted for the difference between consumed 1 and released pNA, and itis postulated that it may correspond to the heterocyclic intermediate I3(Scheme 1a) (See FIG. 24 ). In contrast, consumption of 2 and DPPzformation occurred in parallel with pNA generation (FIG. 17C). ¹H NMRpeaks consistent with the structure of I3 were absent (FIG. 17A). Theseresults demonstrate that isoindole intermediates I3 release aminesrapidly and near-quantitatively.

Given the demonstrated Tz-induced liberation of a reporter molecule(pNA), the potential of BNBDs in a prodrug activation strategy wasevaluated. A 7-acetamide-BNBD doxorubicin prodrug (5) was synthesizedfrom 2d (Scheme 1b). HPLC analysis of the reaction between 5 and PEG-Tz(DMSO, T = 37° C., c(5) = 0.2 mM, c(PEG-Tz) = 1.6 mM) showed rapid andcomplete doxorubicin release (FIG. 18 ).

Tz-triggered drug release was further tested in cell viability assays.Combinations of doxorubicin-prodrug 5 and PEG-Tz caused dose-dependentcytotoxicity in A549 pulmonary adenocarcinoma cells (EC₅₀(5 + 200 µMPEG-Tz) = 96 nM), rivaling that of parent doxorubicin (EC₅₀(Dox) = 88nM, Table 2). Conversely, the prodrug 5 alone was essentially non-toxicin the tested concentration range (EC₅₀(5) > 10 µM; Table 2). Thisoutcome demonstrated that reaction with PEG-Tz efficiently uncageddoxorubicin from 2 and restored its cytotoxicity whereas non-specificdrug release at physiological conditions was minimal. The cytotoxiceffect of 5 was preserved when decreasing the concentration of PEG-Tz to50 µM (EC₅₀(5 + 50 µM PEG-Tz) = 128 nM). Even at the lowest testedconcentration c(PEG-Tz) = 25 µM, which is physiologically attainable,the combination with 5 was >20-fold more toxic than the prodrug alone(Table 2). Tetrazines are rather non-toxic, and in a previous mousestudy, animals showed no adverse reactions to repeated intravenousinjection of doses as high as 1250 µmol/kg. Indeed, control samples withPEG-Tz showed no cytotoxicity even at the highest concentration tested(200 µM). Also,combination of PEG-Tz and 2 resulted in minimal toxicityin the tested concentration range (FIGS. 19A-19B) demonstrating thatcells tolerate isoindole decomposition products well.

TABLE 2 Cytotoxicity of activated prodrug 5, 72 hour incubation at 37°C.^(b) Compounds EC₅₀ (µM) in A549 Cells Doxorubicin 0.088 ± 0.031 5 +PEG-Tz (200 µM) 0.096 ± 0.022 5 + PEG-Tz (100 µM) 0.099 ± 0.029 5 +PEG-Tz (50 µM) 0.128 ± 0.017 5 + PEG-Tz (25 µM) 0.521 ± 0.192 5 >10 2 +PEG-Tz (100 µM) >10 PEG-Tz >200 ^(b) The proliferation assay wasperformed in at least triplicate and EC₅₀ values were derived from thenormalized cell growth.

The stability of 5 in DMSO-PBS (1:1, v/v) was performed as illustratedin FIGS. 23A-23C. Further, the stability of 5 in human serum wasdirectly tested. 5 was inert for 48 h, and no free doxorubicin ordoxorubicin-containing side products were observed by HPLC (FIGS.20A-20D). The quantity of 5 decreased with longer incubation times butno free doxorubicin was detectable (FIGS. 20A-20D). In light of theknown instability of the doxorubicin moiety in serum, it was reasonedthat decomposition of doxorubicin rather than the BNBD linker wasresponsible for the observed effect. To test this hypothesis, theserum-stability of 2 was measured. It was determined that 2 wascompletely stable until the end of the analysis at one week and notraces of pNA were formed. These experiments demonstrated the potentialof BNBDs as prospective chemically-triggered drug release molecules.

The present data demonstrates that BNBDs react rapidly with Tz andgenerate hydrolysis-susceptible heterocycles for the traceless releaseof a drug or reporter molecule. This novel probe design relies onunprecedented self-immolative isoindole/isobenzofuran intermediates forcargo liberation (See FIG. 15 for Scheme 1). The reaction exhibitsfavorable characteristics, including rapid bimolecular reaction,quantitative as well as near-instantaneous payload release, low reagenttoxicity, and exceptional probe stability at physiological conditions.

High stability is a major benefit of BNBDs as drug release molecules. Nobackground liberation of reporter molecules was observed even afterincubating 2 with human serum for a week (FIG. 21 ). Additionally, BNBDderivatives are expected to retain their reactivity whereas TCO-derivedmolecules gradually deactivate by spontaneous trans/cis isomerization.In contrast to the exceptional stability of the BNBD precursors, payloadrelease upon reaction with Tz is rapid and near-quantitative. Thecombination of high BNBD stability and facile drug release will beessential for achieving a high therapeutic index in targeted drugdelivery approaches. This effect was confirmed in cytotoxicity assayswith cultured A549 cells. Although the prodrug 5 alone showed notoxicity in the tested concentration range, it was highly cytotoxic whencombined with Tz at concentrations that were far below doses toleratedin vivo. Also the rate of the reaction of BNBD and Tz compares favorablywith known release designs. The reaction of BNBDs and Tz issignificantly faster than many reported bioorthogonal release reactions,and is of similar magnitude to the reaction of TCO-prodrugs withdimethyltetrazine. Considering the strong observed solvent effect (Table1), it is plausible that the measured rates considerably underestimatethe reaction speed in physiological samples.

Further, the straightforward synthesis of BNBD-release molecules is adistinct advantage of these molecules. In particular, the precursor 2dis accessible in a single step. In contrast, the preparation ofTCO-prodrugs requires multi-step synthesis, including the separation ofthe axial from the equatorial stereoisomer.

The foregoing detailed description describes the invention withreference to specific exemplary embodiments. However, it will beappreciated that various modifications and changes can be made withoutdeparting from the scope of the present invention as set forth in theappended claims. The detailed description and accompanying drawings areto be regarded as merely illustrative, rather than as restrictive, andall such modifications or changes, if any, are intended to fall withinthe scope of the present invention as described and set forth herein.

What is claimed is:
 1. A bioorthogonal molecule, comprising: a moleculehaving a structure according to:

wherein R¹—R⁸ are independently selected from H, a substituted orunsubstituted C₁-C₄ alkyl or alkylene group, COOH, COOR⁹, COR⁹,CONR⁹R¹⁰, CN, CF₃, and SO₂R⁹, where R⁹ and R¹⁰ are independentlyselected from H and a substituted or unsubstituted C₁-C₄ alkyl oralkylene group, with the proviso that one of R³—R⁸ comprises a leavinggroup, and wherein X is O, S, N, SO, SO₂, SR⁺, Se, PO₂ ⁻, or NRR′⁺, andwhere R and R′ are independently selected from H or a substituted orunsubstituted C₁-C₄ alkyl or alkylene group.